Chapter PHYSICOCHEMICALPROFILING INDRUGRESEARCHANDDEVELOPMENT KrisztinaTakács‐Novák Contents 1.1. INTRODUCTION...........................................................................................................................................3 1.2. THEORETICALBACKGROUND..............................................................................................................6 1.2.1. Thephysical‐chemistryofdrugaction................................................................................6 1.2.2. Physicochemicalparameters...................................................................................................8 1.2.2.1. Ionization(pKa).............................................................................................................8 1.2.2.2. Solubility(logS)........................................................................................................17 1.2.2.3. Lipophilicity(logP)..................................................................................................20 1.3. METHODSFORPHYSICOCHEMICALPROFILING.......................................................................25 1.3.1. pKadetermination......................................................................................................................25 1.3.1.1. Potentiometricmethod..........................................................................................26 1.3.1.2. UV/pHtitration..........................................................................................................27 1.3.1.3. Othermethods............................................................................................................28 1.3.1.4. Co‐solventmethod....................................................................................................30 1.3.1.5. Decisiontreeformethodselection...................................................................31 1 Chapter1 1.3.2. logSdetermination....................................................................................................................32 1.3.2.1. Methodsfordeterminationofkineticsolubility..........................................32 1.3.2.2. Methodsfordeterminationofequilibriumsolubility...............................33 1.3.2.2.1. Saturationshake‐flaskmethod(SSF).............................................33 1.3.2.2.2. Potentiometricmethods........................................................................34 1.3.2.2.3. μDISSmethod.............................................................................................35 1.3.2.2.4. Highthroughputmethods....................................................................35 1.3.2.3. Specialapplications..................................................................................................36 1.3.3. logPdetermination....................................................................................................................37 1.3.3.1. Shake‐flask(SF)method........................................................................................37 1.3.3.2. Potentiometricmethod...........................................................................................38 1.3.3.3. IndirectlogPmeasurementmethods..............................................................39 1.3.3.4. Highthroughputmethods.....................................................................................39 1.3.3.5. Decisiontreeformethodselection....................................................................40 1.4. CASESTUDIES............................................................................................................................................41 1.4.1. pKadetermination.......................................................................................................................41 1.4.2. logSdetermination....................................................................................................................44 1.4.3. logPdetermination....................................................................................................................49 1.5. OUTLOOK.....................................................................................................................................................52 Acknowledgement..............................................................................................................................................52 REFERENCES.........................................................................................................................................................52 2 1.1. INTRODUCTION Thepurposeofdrugresearchistodevelopeffective,safe,andhighqualitynew medicines to treat diseases where no drugs or otherwise nonoptimal ones are available.Thisactivityisverycomplex,lengthy,expensive,andrisky.Sincedrug research became industrialized, the highest level of scientific and technological knowledge has been applied during the given era. Fundamentally, the industry usesandputsintothepracticethenewestscientificresultsasearlyaspossible thusdrugresearchitself becomesthedrivingforceforthedevelopment ofnew theories,technologies,andmethods[1]. Takingalookbackatthehistoryofdrugresearch,onecanrecognizeonthelong way of the evolution of the present system some milestones, paradigm‐changes whichresultedinconsiderabledevelopmentinitsage(Figure1.1).Inthe‘60sof thelastcentury,theformerlyusedtraditionalmethods(suchastheextractionof activecompoundsfrommedicinalplants;randomscreening,trial‐errormethod; side‐effect observation; serendipity, etc.) more or less have been replaced or at least extended by the new strategy of rational drug design. Its first application wastheQuantitativeStructure‐ActivityRelationships(QSAR)analysisintroduced by C. Hansch [2] and based on the accumulated knowledge of structure‐activity relationships.Therationaldrugdesignwascompletedwiththeapplicationof3D molecular modeling, theoretical and computational chemistry (Computer Aided DrugDesign,CADD)andprovedtobeamoreeffectivetoolthanpreviousonesin thediscoveryandoptimizationofnewactivemolecules.Theappearanceandfast expansionofhighthroughputscreening(HTS)andcombinatorialchemistryinthe ‘90shavegreatlyenhancedthenumberofactivecompoundsfound[3].Thelatest paradigm‐changewasprovokedbythehumangenomeprojectandtheincreased number of potential targets identified by genomics. However, these changes in the research strategy did not mean that former methods were completely neglected,indeedamajorityofthemareaninherentpartofdrugresearch.Each methodhasitsappropriateuseandimportanceinit. Sincethefirstrecognitionsofstructure‐activityrelationships,medicinalchemists involved in drug research have been always paying outstanding attention to those properties of drugs which determine their pharmacological action. The knowledge of solubility, ionization ability and lipophilicity of drug candidates provides useful information about the expectable pharmacokinetic properties 3 Chapter1 and gives synthetic chemists adequate tools to improve them by modifying the structuralmoietiesofthemolecule[4]. traditional methods rational drug design QSAR 1960 CADD 1980 HTS Combi Chem 1990 genomics proteomics metabonomics 2000 Figure1.1.Strategiesindrugresearch 2010 In the past, however, the main focus of drug research was first devoted almost exclusively to the pharmacodynamic aspects of the biological activity and only laterinthe developmentphasewerethepharmacokineticpropertiesexamined. Thishasledtoahighattritionrateofcompounds.Inthelate‘80sstudiesreport‐ edtwoprominentreasonsofdrugcandidatefailure:thepoorbiopharmaceutical properties (e.g. low bioavailability) and safety. Pharmaceutical companies have made initiatives to shift the physicochemical profiling of compounds earlier in thedrugdiscoveryprocess[5]. Currently, drug research is usually divided into two main phases: (1) discovery phase, which involves the target identification, hit discovery, lead selection and optimization;and(2)developmentphaseinwhichpreclinicalandclinicalstudies are conducted (Figure 1.2). The role and timing of the physicochemical characterization has considerably changed. The new strategy applied since the ‘90s is based on a parallel optimization of efficacy and prognostic profiling of drugability.Thisrequiredanewmentality:tobreakdownthewallbetweenthe discovery and development phases and to migrate from sequentially assessing efficacy and drugability to the parallel process; to evaluate the therapeutic and drug‐likefeaturestogether[6]. Figure1.2.Drugresearchprocess Goodpharmaceuticalproperties,besidestheefficacy,meangoodabsorptionand distribution, chemical and metabolic stability (appropriate bioavailability) and lowtoxicity. 4 Physicochemicalprofilingindrugresearchanddevelopment Foroptimizationofdrug‐likeproperties,physicochemicalparametersaresimple andcheaptoolsintheearlyphaseofdrugresearch.Determinationofproperties relevanttobiologicalactivityofdrugssuchasionization,solubility,lipophilicity andpermeabilityiscalledphysicochemicalprofiling(Kernsetal.2001.)[7]. It is distinguished from the more complex term pharmaceutical profiling which involves the investigation of integrity, stability, metabolic properties (e.g. CYP 450 inhibition), transporter effects and drug‐drug interactions as well (Figure 1.3)[8]. pharmaceutical profiling ionization drug-drug interactions integrity solubility lipophilicity permeability metabolism physicochemical profiling stability transporter effects Figure1.3.Pharmaceuticalprofilingvs.physicochemicalprofiling The present chapter focuses on only three parameters of physicochemical profiling (pKa, log S, and log P) while Chapter 3 is dedicated to the role and determination of membrane permeability. The traditional non‐automated, time‐ andmaterial‐consumingmethodsdevelopedinthepastforphysicochemicalpro‐ filingarenotsuitableindiscoveryforthemeasurementofthedrasticallyincre‐ asednumberofnewchemicalentities(NCE).Nowadays,suchearlyphysicochem‐ icaldeterminationsmustbematerial‐saving,HT,andreasonablyreliable.Several excellent commercial instruments have been developed for this purpose, which areminiaturized,automated,andadaptedtohigh‐throughputtechnologies[9]. Thefirstcomprehensiveoverviewofphysicochemicalprofilingwasreportedby P.Taylorin1990[10].Theprogressivedevelopmentachievedinthenextdecade is surveyed in A. Avdeef’s book: Absorption and Drug Development: Solubility, Permeability and Charge State [11]. This book can be considered as the most competentanddetailedcompilationofadvancedknowledgerequiredbyphysical chemists involved in drug development. Numerous reviews summarized the state‐of‐the‐artofnewHTexperimentaltechniques[12‐15],themostrecentwas 5 Chapter1 publishedbyY.Henchozetal.[16].So,variousliteraturesourcesareavailablefor all who would expand their understanding of physicochemical profiling accordingtotheirneed. Theaimofthischapteristoprovide:(i)aconcisesummaryoftheoreticalback‐ ground; (ii) a comparison of different experimental methods and approaches; (iii) an introduction to ample, useful, and practical examples. The case studies taken from more than 30 years of experiences of the author are intended to providehelptophysicalchemistsintherightmethodselectionandmeasurement ofdifficultmolecules. 1.2. THEORETICALBACKGROUND 1.2.1. Thephysicalchemistryofdrugaction Drug action is a consequence of several chemical and biological processes in whichbindingtothereceptor(pharmacodynamicphase)isessential.Besidesthis however, the pharmacokinetic processes have also fundamental importance in the biological activity. The active ingredient of a drug must separate from the appliedpharmaceuticaldosageform,mustdissolveinbodyfluidsandpermeate throughbiologicalmembranestoreachthereceptorsite.Followingthereceptor response, the active compound dissociating from the binding site generally undergoes metabolism and is excreted from the body. These liberation, absorp‐ tion, distribution, metabolism, excretion (LADME) features are mainly deter‐ mined by the physicochemical properties of drugs, namely by ionization, solu‐ bilityandlipophilicity. Biological membranes are the main physiological permeation barriers to be crossedbydrugs.Structurally,theyhavealipidbilayerresultingfromtheorien‐ tation of amphiprotic lipids (phospholipids, glycolipids, sphyngomyelin) and cholesterolintheaqueousmedium.Thisbilayerhassomeofthepropertiesofa two‐dimensionalfluid(fluid‐mosaicmembrane model)inwhichindividuallipid molecules can diffuse rapidly in the plane of their monolayer (lateral mobility) butcannoteasilypasstotheothermonolayer.Animportantobservationisthat phospholipids are asymmetrically distributed in the membrane. Generally, the outer (extracellular) half of the bilayer comprises mainly zwitterionic lipids (phosphatidylcholine andphosphatidylethanolamine),whereastheinner(intra‐ cellular) part contains negatively charged lipids (e.g., phosphatidylserine). Dif‐ ferent proteins that induce transporter, signal transduction, or metabolic func‐ tionsareintegratedintothelipidbilayer[17,18].Thebiologicalmembranesare apolarbarriers,wheretherelativepermittivityinsideisextremelylow(ε~2).It has long been assumed that most drugs use transcellular transport and pass these barriers by passive diffusion which is favorable only for unionized, lipophiliccompounds.Thereareseveralothermechanismsofpermeation.Active transport is ligand‐mediated by different transporters for compounds. Paracel‐ lular permeation exists between the cells for smaller, more polar compounds. 6 Physicochemicalprofilingindrugresearchanddevelopment Some compounds are transported by endocytosis, when the molecules are engulfed by the membrane and move through the cell in these membrane‐ enclosed vesicles. For further detailed information, the reader is encouraged to reviewspecializedresources[18,19]. ThepH‐partitionhypothesis[20]providesagoodmodelforthepassivetransport ofionizablemoleculeswithsufficientlipophilicity.Figure1.4showsaschematic representation of the transport of a basic (B) (e.g. papaverine, chlorpromazine, etc.)andanacidic(HA)(e.g.acetylsalicylicacid,ibuprofen,etc.)molecule.Inthe extracellularaqueousmedium,theratioofionized([BH+]or[A‐])andunionized ([B]or[HA])formsisdependentontheactualpHofthegivencompartmentand the pKa of the compound. The uncharged, neutral species has much higher lipophilicitythanitscharged(ionic)form,thusitcanpermeatethroughthelipid membraneevenifbeing presentas aminorcomponent.Inmedicinalchemistry thisspeciesiscalledthe“transportform”.Enteringintotheintracellularaqueous phase,anotherionizationprocesstakesplaceresultingintheionizedformagain whichgenerallyinteractswiththetargetandisreferredtoasthe“receptorform”. Theamountofthetransportformpresentatthemembranesurfacedependson itssolubility.Moleculesmustbeinsolutioninordertodiffuseintothemembra‐ nes,howeverlowsolubilitycanbealimitingfactorofpermeation.Permeability asadeterminantkineticparameteroftransportisdiscussedinChapter3. AH pKa - A +H + + B+H + pKa extracellular BH intracellular AH - + + A +H B+H + BH receptor Figure1.4.Transportandreceptorformsofanacidandabase The concept derived from the pH‐partition theory that “only neutral molecules permeatemembranes”startedtobequestionedfromthemid‘90sbecauseofan increasing body of experimental evidence supporting ion‐partitioning into artificialmembraneslikeliposomes[21].Thiswasinterpretedwithelectrostatic interactions and hydrogen bonding between the charged group of compounds 7 Chapter1 and ionized polar head group of phospholipids in the “pH priston model” [22]. Recently, S. Krämer and coworkers [23] reviewed the mechanisms underlying lipid bilayer permeation. They proposed a kinetic “flip‐flop model” based on a three‐stepmechanism,namelythepartitioningintoonelipidlayer,translocation (flip‐flop) to the opposite lipid layer and partitioning into the aqueous phase. Accordingtothismodel,thepermeationofachargedspeciescouldbetheresult of occasionally occurring trans‐membrane translocation of charged compounds. It was concluded that membrane permeation is more complex than expected fromasimplediffusionmodelandpH‐partitionhypothesis. Anotherpossiblemechanismoftransportforionized,hydrophiliccompoundsis carrier‐mediated active transport. The increasing number of different uptake transportersdiscoveredinthepast15yearshighlightstheimportanceoftherole of active transport in membrane permeation of drugs which may be under‐ estimated.Theirphysiologicalfunctionistodeliverthenecessarynutrientsand other endogenous biochemical compounds having low lipophilicity for passive diffusion to the cell. Several drugs were found to be the substrate of different specific transporters like oligopeptide (PEPT1: captopril, enalapril, ampicillin, acyclovir),organicanion(OATP1:fexofenadine,enalapril,temocaprilat),organic cation (OCT1: metformin, famotidin), or nucleoside, etc. [18,24]. The efflux transporters (P‐glycoprotein, P‐gp; breast cancer resistance protein, BCRP; multidrugresistanceprotein,MRP2)assistinthemovementofcompoundsoutof thecellastheyprotectthecell frompotentially toxicxenobiotics.Thisoutward transporthasanegativeeffectonthepharmacokineticsofsomecompounds.The activity of efflux transporters is very intensive in the blood‐brain barrier and sometumorcellsresultinginmultidrugresistance.Bindingtothetransportersis determined by the chemical structure of compound. Similar moieties to the natural substrate, a large number of H‐bond acceptors (N + O atoms), and high molecularweight(Mw>400)appeartoincreasethelikelihoodofP‐gpefflux[25]. Physicochemical properties influencing the fate of a drug in the body are described by the thermodynamic equilibrium constants. Below, we summarize thefundamentalsofpKa,logS,andlogPterms. 1.2.2. Physicochemicalparameters 1.2.2.1. Ionization(pKa) Drugs are multifunctional compounds. A great majority of them contain one or moreionizable(acidicorbasic)functionalgroups.Inaqueoussolutions,ionizable compoundsexistindifferentionization(chargedoruncharged)statesdepending ontheirstrengthofacidityorbasicityandthepHofthesolution. Definitions,terms Theionizationconstant(oraciddissociationconstant),Ka,isusedtocharacterize the acid‐base chemistry of a molecule generally expressed as a negative loga‐ rithm:‐logKa=pKa.Inmedicinalchemistry,itiscommontousepKaforbothacids 8 Physicochemicalprofilingindrugresearchanddevelopment andbases.Inaqueoussolutions,thepKascalespans from0to14. Thestronger the acid, the lower is its pKa value. The opposite is true for bases; a higher pKa valuemeansstrongerbasicity[26]. increasing acidity 0 14 CF3COOH 0.23 salicylic diclofenac acid 3.99 2.88 phenobarbital acetaminophen 7.49 9.63 increasing basicity 0 caffeine benzocaine aminophenazone 0.60 2.39 14 debrisoquine 13.01 5.06 papaverine 6.39 amlodipine 9.26 propranolol 9.54 ephedrine 9.60 atropine 9.84 Figure1.5.ThepKascaleinaqueousmedium Some examples for the most frequently occurring acidic and basic functional groupsindrugsarelistedinTables1.1and1.2. Equations1.1‐1.4showtheionizationequilibriaandtherelevantthermodynamic ionization constants using general symbols: HA for acid, B for base, XH for diproticampholytemolecule. ‐ + HA A +H K a pK a pH log + [HA] [A ] [A ][H ] HA BH B+H K a (1.1a,1.1b) (1.1c) [B][H ] [BH ] (1.2a,1.2b) 9 Chapter1 Table1.1.Someimportantacidicfunctionalgroupsindrugs group name pKa example(pKa) CH3 sulphonicacid 0‐1 O C OH carboxyl 2‐7 O enol C C OH N N CH3 OH O C C C N H N S CH3 O O 2‐6 N N piroxicam (2.33) CH3 CH3 H3C tetrazole metamizole benzoicacid (3.98) N HOOC O N N CH3 OH C O H N N HO3SCH2 O S OH O valsartan (4.8) 4‐5 N N N N H O O S N C O H O NH S O sulphonimide 5‐6 O O S N C N C4 H O H H3C N‐aryl‐ ‐sulphonamide 6‐8 O N S N O H N H2N O lactam CH3 sulfadimidine (7.49) phenol C HN 7‐8 9‐11 CH3 O OH CH3 CH3 NH tolbutamide (5.3) O C O barbital(7.9) NH OH HN O C CH3 acetaminophen (9.63) CH3 SH 10 thiol 8‐11 HS O N COOH captopril(9.8) Physicochemicalprofilingindrugresearchanddevelopment Table1.2.Someimportantbasicfunctionalgroupsindrugs group name pKa example(pKa) NH HN C HN guanidine N 13‐14 NH NH2 debrisoquine(13.0) OH HO aliphatic primary amine NH 2 NH2 HO noradrenaline(8.5) OH aliphatic secondary amine NH NH 8‐11 CH3 CH3 ephedrine(9.6) CH3 O C N H CH3 aliphatic tertiary amine N CH3 N CH3 lidocaine(7.9) aromatic primary amine NH R aromatic secondary amine NH2 O C O H2N CH3 benzocaine(2.4) CH3 HN 2‐5 O C O tetracaine(2.4) CH3 N CH3 Cl N R R' aromatic tertiary amine N N CH3 N CH3 chloropyramine(2.0) 11 Chapter1 pK a pH log [BH ] [B] (1.2c) XH2 XH+H+ K a1 XH X ‐ +H + K a2 pK a1 pH log [XH][H ] [XH2 ] (1.3a,1.3b) [X‐ ][H ] XH (1.4a,1.4b) [XH2 ] [XH] pK a2 pH log [XH] [X ] (1.3c,1.4c) Incertainresearcharticles,preferenceisgiventotheuseoftheionizationrather than the proton association process and the term protonation constant, Kp, particularly in coordination chemistry [27]. The relationship between them is reciprocal where Ka= 1/Kp, or pKa = log Kp. For a monoprotic compound this relationshipisevident,butmaynotbeclearregardingmoleculeswithmorethan one ionizable group. Below, we describe the ionization processes of a triprotic compound (like amoxicillin) from both points of view: dissociation (molecule releasestheproton)andassociation(moleculegainstheproton). O HOOC N H3C H3C N H S OH O NH2 Dissociation K p1 ‐ X 2‐ +H+ XH K a1 K p3 K a2 (1.6a,1.6b) K p3 (1.7a,1.7b) + XH2 +H+ XH3 K a3 2‐ + XH ‐ X +H 1 Kp2 K a3 1 K p1 pKa1=logKp3pKa2=logKp2pKa3=logKp1 12 (1.5a,1.5b) K p2 XH‐ +H+ XH 2 K a2 + XH2 XH +H 1 Protonation + XH3+ XH2 +H K a1 log Kp1= 9.6 log Kp2= 7.4 log Kp3= 2.4 (1.8a,1.8b,1.8c) (1.9a,1.9b,1.9c) Physicochemicalprofilingindrugresearchanddevelopment Ionizationmicroconstants Theequilibriaabovecharacterizethedissociation/protonation ofthemoleculeat the molecular level, so called macroscopic level, using ionization macroconstants. Ionization macroconstants quantitate the overall acidity/basicity of the molecule, butcannotbeassignedtoindividualprotonbindingsitesofmultiproticmolecules. Ionizationmicroconstantsarethetermswhichdescribetheprotonbindingability oftheindividualfunctionalgroupsandareusefulincalculatingthepH‐dependent concentrationsofmicrospecies(namedmicrospeciation)[28].Inthepastdecade, themicrospeciationofseveraldrugmoleculeswaspublished[e.g.29‐31]. The macroscopic and microscopic protonation scheme of a diprotic molecule usingnorfloxacinasamodelisshowninFigure1.6.Forsimplicity,Kdenotesthe protonation macroconstants and k is used for microconstants. The superscript denotes the functional group is protonating in a given process, the subscript (if any) shows the already protonated group and N and C refer to the piperazine nitrogen and the carboxylate group, respectively. There are two possible alter‐ nativeroutesofprotonation.Fromthemostbasicanionicform(X‐)thecarboxy‐ late group first accepts a proton resulting in the chargeless (XHo) form, then a secondary amine group protonates producing the cation (XH2+) (lower route). The other pathway of protonation is conducted through the formation of a zwitterion(XH±)duetotheprotonationof an aminogroup first.Thechargeless and zwitterionic forms are chemically different microspecies (they bear the proton on different binding sites) having the same stochiometric composition (oneprotonisaccepted),sotheyareprotonationisomers. O - F k H H N N k C N O CH3 H - F N N + N O COO COO N F N CH3 k C - K1 N N N + COOH N H X N O F COOH H k N CH 3 H N C CH3 XH K2 XH2 + ß2 Figure1.6.Theprotonationmacro‐andmicro‐equilibriaofnorfloxacin 13 Chapter1 Therelationshipsbetweenthemacro‐andmicroconstantsarethefollowing: K1=kC+kNK1K2= k C k CN = k N kNC 1 K2 1 N C k 1 kNC (1.10a,1.10b) (1.10c) Once macro‐ and microconstants are known, the mole fraction of each species can readily be calculated and the pH‐dependent distribution of macro‐ and microspecies can be constructed. Figure 1.7 shows the distribution of different protonationformsofnorfloxacinagainstthepHandindicatesthepredominance of the zwitterionic form over the chargeless microspecies. However, it is also visiblethattheirconcentrationattheiso‐electricpointpHiscommensurableand bothformsarepresentinasignificantamount. 100 80 % 60 40 20 0 2 4 6 8 10 12 pH Figure1.7.Distributioncurveofthe4microspeciesofnorfloxacinasafunctionofpH The microspeciation of a triprotic molecule [32,33] is more complicated, conta‐ ining8microspecies.Thetotalprotonationprocesscanbedepictedby12micro‐ constantsasdemonstratedincaseofamoxicillininFigure1.8.TheO,N,Csub‐or superscripts of the k microconstant refer to the three proton binding sites, namelyphenolate,amino,andcarboxylategroups,respectively.Therelationships betweenthemacro‐andmicroconstantsarethefollowing: 14 K 1 k O k N k C (1.11) K1K2 kOkON kOkOC kNkNC kNkNO kCkCO kCkCN (1.12) C C K 1 K 2 K 3 k O kON kO,N k N kNO kO,N ........ (1.13) Physicochemicalprofilingindrugresearchanddevelopment The theory and practice of proton microspeciation based on NMR‐pH titration anddataintheliteratureoncompletemicrospeciationofsmallligandsincluding drugshaverecentlybeensurveyed[34]. HO HO NH2 O N O O - - OOC S CH3 H3C O C NH2 C k N ,O HO HO N H3 C NH2 NH3 O N k NH O OOC O kN kO O - N CH3 H3 C NH O S OOC k - NH3 O NH O - N kO O NH O N S - CH3 OOC - N S CH3 H3 C NH O - O O NH N S H3 C CH3 O k C kN NH3 NH2 O N HOOC S H3C XH - O N NH O 2- NH3 k N,C C X HOOC CH3 H3 C O O S HOOC O kC N k O ,C kC NH O N CH3 HOOC H3 C XH2 S CH3 XH3 + Figure1.8.Protonationmacro‐andmicro‐equilibriaoftriproticamoxicillin Temperatureandionicstrength The ionization constant as a thermodynamic parameter is temperature‐ dependent.FortheprecisedeterminationofpKa,experimentsmustbeconducted undercontrolledconstanttemperature.Inpractice,thecommonreferencevalue is 25 °C and only few data are available measured at 37 °C. The change in pKa uponanincreaseoftemperaturefrom25°Cuptothephysiologicaltemperature of37°Cisdependentonthegiven molecule.Generallythechangeinthe pKaof acids is less, while bases are more sensitive to temperature change [26]. The approximate average value of temperature dependence is known as δpKa/δT: 0.02‐0.03,whichmeans0.24‐0.36ΔpKavaluesbetween25and37°C.IfthepKaof acompoundfallsintothepHrange1.5‐8(thepHgradientpresentinthehuman gastrointestinaltract),thenevenarelativelysmalldifferencemayleadtopoorin 15 Chapter1 vitro‐in vivo correlations. For a better interpretation of the cellular transport mechanismofsuchmolecules,thebiorelevantpKavalueisparticularlyuseful.A prediction method for this value based on a 2D structure and pKa at 25 °C was proposedveryrecently[35]. The ionic strength of the medium also affects the pKa value. It is common to measure at constant ionic medium, generally at I = 0.15 M adjusted by KCl or NaCl corresponding to the physiological level. Frequently, a different ionic mediumisusedordatacalculatedtozeroionicstrengthusingtheDebye‐Hückel theoryarealsopublished,thusitisalwaysnecessarytoreporttheionicstrength andtemperatureofapKameasurement. ImportanceofpKainmedicinalchemistry ThedegreeofionizationatagivenpHcanbecalculatedoncethepKaisknown.As aruleofthumb,atpH=pKa50%ofthecompoundisionizedand50%isinthe unionizedform,whileatpH=pKa±2predominanceofonespeciesbecomes99 %.Forexample,anacidispresentin99%atpH=pKa‐2asunionized(HA)and atpH=pKa+2asionized(A−)(theoppositecaseappliestoabase). The ionization state determines the transport properties, thus its precise calculation allows the estimation of ADME features. With the knowledge of the pKa value, the proportion of the transport form can be calculated at any physiologically important pH values. Regarding ampholyte compounds, the pKa valuesareusefultocalculatetheiso‐electricpointorthepHatwhichamolecule has the lowest solubility and highest lipophilicity. Since solubility, lipophilicity, andpermeabilityarepH‐dependentproperties,thepKavalueofanewmolecule mustbedeterminedinadvancetothelogS,logPandpermeabilitymeasurement. Ionic interactions play a fundamental role in the receptor binding of ionizable molecules. An ionic bond is the strongest non‐covalent binding type. The electrostatic attraction of opposite charges directs the molecule to the receptor surfaceandelectrostaticcomplementaritywiththereceptor isaprerequisiteof anydrugaction. Antiarrhythmicdrugs(classI:Na+‐channelantagonists)serveasagoodexample ofhowpKaaffectsdrugaction.ThesedrugsareweakbaseswithmosthavingpKa values ranging from 7.5 ‐ 9.5. At the physiological pH of 7.4 they exist in an equilibrium mixture consisting of both the free base (B) and protonated (BH+) cationicform.IncompoundswiththepKa>9(likeprocainamide,mexiletine,pro‐ pafenone),thepresenceofthereceptorformexceeds90%whichisfavorablefor thebindingtothesodium‐channel.However,forcompoundsinwhichthepKa<8 (likequinidine,lidocaine)thisratioismuchlessfavorable(Table1.3).Lidocaine (pKa = 7.96) has a stronger electrophysiologic effect in ischemic than normal myocardialtissue.Thispotentiationhas,inpart,beenattributedtotheincrease in H+ concentration (lower pH) within the ischemic areas of the heart. Acidosis increasestheportionofreceptorformofthedrug(Table1.3)andconsequently theproportionofNa+‐channelsoccupiedbytheBH+oflidocaine[36]. 16 Physicochemicalprofilingindrugresearchanddevelopment Table1.3.IonizationstateofantiarrhythmicclassIdrugsatnormalandischemictissue compound procainamide pH=7.4(normaltissue) pH=6.4(ischemictissue) BH+,% B,% BH+,% B,% 98.4 1.6 99.9 0.1 mexiletin 98.2 1.8 99.8 0.2 quinidine 76.0 24.0 99.0 1.0 lidocaine 78.4 21.6 97.3 2.7 1.2.2.2. Solubility(logS) Solubility is a molecular property which determines the maximal concentration ofasoluteinagivensolvent.Theaqueoussolubilitydependsonthepolarityofa molecule and varies with the pH for ionizable compounds. Solubility can be described by different parameters and a vast variety of terms and symbols are usedtoexpressthesolubilitydataofcompounds,hencebelowwesummarizethe mostcommonbasicdefinitions. Definitions,terms Equilibrium(orthermodynamic)solubility(S)istheconcentrationofacompound inasaturatedsolutionwhenasolidispresentandthesolutionandsolidareat equilibrium. This value is constant at a given pressure and temperature and characteristicforagivencompound. Forionizablemolecules,furthertermsaredistinguished.Intrinsicsolubility(S0)is theequilibriumsolubilityofafreeacid(HA)orfreebase(B)formofanionizable compound at a pH where it is fully unionized. With respect to ampholytes, this referstotheneutral(chargeless)form(XH)whichexistsattheiso‐electric(i.e.) point pH. Effective solubility (SpH) is the equilibrium solubility of an ionizable compoundatapHwherebothunionizedandionizedformsarepresent.Itisalso denotedasapparent(ortotal)solubilityanddefinedataparticularpHasthesum oftheconcentrationsofallcompoundspeciesdissolvedinanaqueoussolution. Thesolubilityofsaltformofanionizablecompound(Ssalt)canbederivedfromthe solubilityproduct(Ksp).Foramonoproticacidorbase: S salt K sp where, Ksp = [A−] [Y+] for an acid and Ksp = [BH+] [X−] for a base, Y+ and X− representthecounter‐ioninthesalt. Recently,anewtermcalledkineticsolubility(SAPP)wasintroducedinearlydrug discovery.Itistheconcentration of asolutionof anexaminedcompound atthe moment when the first precipitation of the solid is observed in an experiment whereasmallvolumeof10‐20μg/mldimethylsulfoxide(DMSO)stocksolution is added to aqueous buffer. This parameter is not a thermodynamic physico‐ chemical constant because the system does not reach an equilibrium state. Generally,SAPPishigherthantheequilibriumsolubilitysincethereisnoneedto overcome the crystal lattice forces by aqueous solvent once the compound has 17 Chapter1 been dissolved in DMSO. Kinetic solubility data are mainly used for ranking the moleculesintheearlystagesofdiscoveryandcannotreplacethedetermination ofthetrueequilibriumconstantlaterinthedevelopmentphase. Theabovesolubilityparameterscanbeexpressedinvariousconcentrationterms like:g/100ml;g/ml;mg/ml;μg/mlormol/L(M);mmol/L(mM); μmol/L (μM), etc.Forbettercomparability,thelogarithmofsolubilityterm(logS)isfrequently usedandcanbeobtainedfromMorμMconcentration.Preferenceforthe–logS term is found in the literature in order to avoid negative numbers for low solubility compounds. However, it may be somewhat confusing because the aforementionedtermyieldshighervaluesmeaninglowersolubility. Solubility is affected by many factors, such as temperature, pressure, pH, ionic strength of aqueous media, purity of a sample, crystal form, particle size, poly‐ morphism,etc.Theeffectofthesefactorshavebeencomprehensivelydiscussed inclassic[37,38]andnewbooks[11,18].Here,wefocusonly onthepHdepen‐ dencyofsolubility. Solubility‐pHprofile ThesolubilityofionizablecompoundsvarieswiththepH.Theyaremoresoluble in the charged than in the unionized form. When a molecule exists only in the monomer state, its pH‐dependent equilibrium solubility is derived from the Henderson‐Hasselbalch (HH) equations (Equations 1.1c‐1.4c). The HH relation‐ shipforamonovalentacid,base,and(diprotic)ampholytemoleculecanbederi‐ ved from solubility and ionization equilibria as follows where, by convention [HA(s)]=[B(s)]=[XH(s)]=1, and [A−], [BH+], [X−], [XH2+] are expressed using Equations1.1b,1.2b,1.3b. acid: HA(s)⇌HA S0 (1.14) [HA] HA [HA(s)] (1.15) S pH = A + HA S pH (1.16) K a [HA] HA [H ] (1.17) K S pH HA a 1 [H ] S pH S0 10(‐pK a pH) 1 (1.18) log S pH log S0 log(1 10(pH‐pK a ) ) 18 (1.19) (1.20) Physicochemicalprofilingindrugresearchanddevelopment base: B(S) B S0 [B] B [B(s)] (1.21) (1.22) ... log S pH log S0 log(1 10(pK a ‐pH) ) (1.23) diproticampholyte: HX(s) HX S0 [XH] XH [XH(s)] (1.24) (1.25) S pH = X + XH + XH2+ ... log S pH log S0 log(1 10(pK a1 ‐pH) 10(pH‐pK a2 ) ) (1.26) Figure1.9showsthecharacteristicsolubility‐pHprofile(aplotoflogSpHvs.pH) foranacid(a),base(b),anddiproticampholyte(c). Figure1.9.Solubility‐pHprofileof(a)anacid,(b)abaseandc)adiproticampholyte 19 Chapter1 The HH relationship can be used to predict the pH‐dependent equilibrium solubilityofdrugswhenthepKaandlogS0valuesofacompoundareknown.Itis a frequent practice in drug research to convert the experimentally measured intrinsicsolubilityvaluetoequilibriumsolubilityataphysiologicalrelevantpHin ordertoestimateitsexpectedbehavior. The validity of the HH relationship has been widely investigated and certain deviations were found [39,40]. They were interpreted with the influence of different molecular interactions such as aggregation and micelle formation [39,41]. Recently, a revisit of the HH relationship concerning organic bases confirmedthevalidityprovidedifhighlyprecisepKaandlogS0valueswereused foritsgeneration[42](seealsoSection1.4.2). ImportanceoflogSinmedicinalchemistry The aqueous solubility of compounds receives considerable attention in drug development, because this is a key molecular property for the gastrointestinal absorption of orally administered drugs. Further on, in biological activity tests compoundsmustbeinsolutionotherwisefalse,erroneousdatacanbeobtained. Lowsolubilityisdetrimentalfrombothpharmacokineticandpharmacodynamic points of view. Determination of aqueous solubility is an inevitable part of physicochemical profiling in drug research. Its importance has grown since the BiopharmaceuticalClassificationSystem(BCS)wasfirstproposedbyG.Amidon in 1995 [43]. This classification uses four classes to categorize drugs based on their solubility and intestinal permeability (class 1: high solubility + high per‐ meability;class2:lowsolubility+highpermeability;class3:highsolubility+low permeability; class 4: low solubility + low permeability). For class 1 molecules, the rate‐limiting factor of intestinal absorption is the rate of dissolution, low solubilityinclass2molecules,whilelowpermeabilityinclass3israte‐limiting. In class 4, both properties are unfavorable for oral administration, and no in vitro‐invivocorrelationcanbeexpected. To improve the in vitro‐in vivo correlation, the measurement of solubility is recommended for biomimetic media as well. There is growing evidence that in theintestine,thepresenceofbileacidsandothercomponentssuchaslipidscan alter (usually increase) the intrinsic solubility of (lipophilic) compounds. Two physiologically relevant media developed by Dressmann et al. [44] are used. These are the fasted‐state simulated intestinal fluid (FaSSIF) and the fed‐state simulated intestinal fluid (FeSSIF) having pH 6.5 and 5.0, respectively, and containdifferentamountsofsodiumtaurocholate,lecithineandsalts[44]. 1.2.2.3. Lipophilicity(logP) Themorefundamentalpropertygoverningthefateofadruginthebodyisundo‐ ubtedlythelipophilicity.Thismolecularpropertyrepresentstheaffinityofamo‐ leculeforalipophilicenvironment.Itismostcommonlydescribedbythelogari‐ thmofpartitioncoefficient(logP)betweentwoimmisciblesolvents,oneisanor‐ ganicapolar(e.g.octanol)andtheotheranaqueouspolar(buffersolution)[45]. 20 Physicochemicalprofilingindrugresearchanddevelopment BesidesP,othersymbolshavebeenusedintheliteraturesuchasPow,Kow,Kp,PC, etc.,however,weusetheterminologywidelyacceptedinmedicinalchemistry. Two types of partition parameters are distinguished: the true partition coef‐ ficient (P) and the distribution coefficient (D or in older literature Papp). Their definitionandrelationshiparebrieflysummarizedbelow. Definitions,terms Thetruepartitioncoefficient(accordingtotheNernstlaw)referstothepartition of a single electrical species, and is expressed as an equilibrium concentration ratioofthesamemolecularforminbothphasesofthesolventsystem.Thisvalue is constant at a given temperature and pressure, independent of the pH and characteristicforthemolecule.Itcanbederivedfortheneutral,monomericform ofacompound(logPN)(Equation1.27)andtheoreticallycanbealsodefinedfor the partition of an ionic form (log PI) (Equation 1.28), but later the value has ordersofmagnitudelowerandinmostofcasescanbepracticallyneglected. PN [unionizedform]octanol [unionizedform]water PN [HA]o [HA]w PI PN (1.27) [B]o [XH]o N P [B]w [XH]w (1.27a-c) [chargedspecies]octanol [chargedspecies]water (1.28) [A‐ ]o [BH ]o [X‐ ]o [XH2 ]o I I I P ‐ P P ‐ P [A ]w [BH ]w [X ]w [XH2 ]w I (1.28a-d) The distribution coefficient of an ionizable compound refers to all species that arepresentinthesolution(Equation1.29).SinceitisapH‐dependentterm,the pHmustbespecifiedasDpH. DpH [unionized ionizedspecies]octanol [unionized ionizedspecies]water (1.29) For monoprotic acid and base: DpH [HA]o [A ]o [HA]w [A ]w pH D [B]o [BH ]o [B]w [BH ]w (1.29a‐b) Fordiproticampholyte: DpH [X ]o [XH]o [XH2 ]o [X ]w [XH]w [XH2 ]w (1.29c) 21 Chapter1 with the assumption that the concentration of the ionic forms in the organic phase is much less than that of the neutral forms (e.g., [A−]o << [HA]o and [BH+]o<< [B]o, etc.) and upon substituting the aqueous equilibrium concentra‐ tionsfromEquations1.1b,1.2b,and1.3b,therelationshipsbetweenPandDcan be obtained. For simple molecules, these relationships are given below (Equations 1.30a‐c) while interactions between more complicated multiprotic compoundscanbefoundintheliterature[46]. Formonoproticacid: log P N log DpH log(1 10(pH pK a ) ) (1.30a) Formonoproticbase: log P N log DpH log(1 10(pK a pH) ) (1.30b) Fordiproticampholyte: log P N log DpH log(1 10(pK a1 pH) 10(pH‐pK a2 ) ) (1.30c) Partitionmicroconstants Similarlytoionizationmicroconstants,micro‐logP(denotedaslogp)ofagiven microspecies of multiprotic compounds can also be defined [46]. This has particular significance in the case of ampholyte compounds where the most lipophilicspecies,theneutral(XH)form,isacompositefromzwitterionic(XH±) andchargeless(XH0)microspecies.Iftheyarepresentinsolutioninacommen‐ surableamount(e.g.,norfloxacininFigure1.7)thentheexclusivepartitioningof thechargelessformcanbeexpectedintothelipophilicphaseandmicro‐logPof XH0 microspecies may be the relevant lipophilicity parameter. Its calculation requires knowledge of the log D at iso‐electric pH value, log Di.e.pH, and the protonationmicroconstants(kC, kCN , k NC )aspreviouslypublished[47]. kN 1 log p0 log D i.e.pH log 1 C + CC kCN H+ k H kN (1.31) Lipophilicity‐pHprofile The plot of log DpH against the pH (lipophilicity‐pH profile) of a compound (Equations1.30a‐c) can be derived from the HH relationships (Equations 1.1c‐ 1.4c),providedthatthereisnoion‐pairpartitioninvolvedintheprocess.Ifsuch ion‐pairpartitionexists,theprofilesshowaplateauatlogDofvalues3‐4orders lower(foracidsathighpH,forbasesatlowpH)thanasindicatedinFigure1.10. Thelipophilicity‐pHprofilesareusefultoestimatetheeffectivelipophilicityofa compound at physiologically relevant pH values and widely used in medicinal chemistry. 22 Physicochemicalprofilingindrugresearchanddevelopment Figure1.10.Lipophilicity‐pHprofileof(a)anacid,(b)abase, and(c)adiproticampholyte SolventsystemsforlogP The widely accepted reference solvent system for log P measurement is octanol/waterproposedfirstbyHansch[45].Inthissystem,thetwophasesare isotropic.Abuffersolutionservesastheaqueousphaseandn‐octanolisusedasa typical H‐bond donor and acceptor organic solvent. This system is thought to modeltheessentialpropertiesofgeneralbiologicalmembranes.Thestructureof water‐saturated octanol became better understood in the ‘90s [48]. Inverted micellaraggregatesareformedwherewaterclustersaresurroundedbyabout16 molecules of octanol, with the polar OH groups pointing to the clusters and intertwined in a hydrogen‐bonding network. The aliphatic tails form a hydro‐ carbon region with properties not too different from the hydrocarbon core of bilayers.Obviously,theoctanol/watersystemcannotbeauniversalmodelforall types of membranes. In the past two decades, partition solvents other than octanol have been explored. Leahy et al. [49] proposed the “critical quartet” system consisting of octanol/water, chloroform/water, alkane/water and pro‐ pyleneglycoldipelargonate(PGDP)/waterforthegeneralmodelingofmembra‐ nes.Later,1,2‐dichloroethane(DCE)andcyclohexanewerefoundusefulorganic solvents.OncelogPhasbeenmeasuredbothinalkane/waterandoctanol/water systems,theΔlogP(logPoctanol–logPalkane)canbecalculated,andusedasasimple parameterfortheH‐bondformationabilityofacompound. Recently, anisotropic systems such as liposomes (vesicles formed from phospholipidbilayers)wereincreasinglyusedtomodelmembranepartitioning. 23 Chapter1 Liposome/water log P values are considered as log Pmem (membrane partition). AnaccumulationoflogPmemdatashowasignificantlyhigherpartitioningofionic forms. Generally, charged species partition into membranes about 100 times more strongly than into octanol. The theory and practice of liposome/water lipophilicitywerereviewed[5,50,51]. ImportanceoflogPinmedicinalchemistry The log P is the oldest and most traditional physicochemical parameter used in medicinal chemistry. Lipophilicity is implicated in numerous biological events (such as transport, receptor binding via hydrophobic interactions, metabolic processes, storage in fat tissues, etc.). The log P value – concerning its infor‐ mationcontent‐ismuchmorethanasimplenumber,becausethesamemolecul‐ ar interactions which exist between the compound and the biological environ‐ ment results in this value. At the same time, log P is very easy to handle by chemistsforcomparisonofmoleculeswithdifferentlipophilicityandestimating theexpectedtransportbehaviorinthebody. Among the properties suggested by Lipinski, (known as “rule of 5”) one of the criteria for drug‐likeness is that log P should be below 5 [52]. It seems to be a reasonableconceptsince90%ofmarketeddrugshavealogPvalueintherange of 0 – 5 (see Figure 1.11). From hydrophilic compounds (log P < 0) good solubility,butpoorabsorptionfromtheGItractcanbeexpectedexceptforthose whichhaveactivetransport(suchasforexampleascorbicacid).Compoundswith moderate lipophilicity (log P between 0 and 3) are optimal for oral administration due to a good balance of solubility and permeability. For good blood‐brainbarrier(BBB)penetration,theoptimallogPvalueisabout2. log P scale … … -2 0 2 4 8 6 drugs 90% HO OH O HO O OH H 3C + CH 3 N Br- I S O O OH O O N Cl CH 3 N CH 3 N CH 3 CH 3 I O CH 3 ascorbic acid methylhomatropine -bromide chlorpromazine amiodarone log P: P: -1.85 absorbs by active transport log P: -1.68 no absorption no BBB penetration log P: 5.34 good oral absorption good BBB penetration log P: 7.57 storage t1/2: 25-30 days Figure1.11.ThelogPscaleofdrugs Highly lipophilic compounds (logP>5) are sparingly soluble in aqueous compartments,tendtoaccumulateinlipoidalpartsandarealsomoresensitiveto 24 Physicochemicalprofilingindrugresearchanddevelopment metabolism. Extremely high lipophilicity may lead to strange pharmacokinetics, for example, amiodarone has log P=7.37 and half‐life t1/2: 25‐30 days(!) (Figure1.11).First,in1987Hanschcalledattentiontothedangerofexceedingly high lipophilic drug candidates and proposed the “minimal hydrophobicity” concept for the design of new compounds [53]. Since then, the unfavorable tendencyofhighlylipophilicdrugproductionhasnotstopped,asnewmolecules indrugresearcharegettingmorelipophilicandlesswater‐soluble[54]. 1.3. METHODSFORPHYSICOCHEMICALPROFILING Demands set up to the methods for physicochemical profiling are different in variousphasesofdrugresearch.Inthediscoveryphase,thedrasticallyincreased numberofNCEsproducedbycombinatorialchemistryrequireshighthroughput (HT), material saving, automated approaches, while less emphasis is placed on precision. A method for physicochemical profiling is considered HT when its capacity exceeds the measurement of 50 compounds/day [7]. Later, in the development phase reliable, precise data are needed which is why accuracy is moreimportantandnotthespeedofthemethod. Thissubchapterisdedicatedtoexperimentalmethodsusedforthemeasurement of pKa, log P, and log S values and comparison of their capacity, accuracy, time, andmaterialdemand(seeTables1.4‐1.6).Wefocusheremainlyonthepractical aspects of their application, while the detailed theoretical background of the methodsisoutofthescopeofthisreview.Forthispurpose,excellentbasicbooks arerecommendedtoreaders[11,18,26,37]. 1.3.1. pKadetermination PotentiometryandUVspectroscopyarethecommonlyusedstandardmethodsof pKa determination. Due to its simplicity and precision, potentiometry is the methodofchoiceoncetheaqueoussolubilityofacompoundreachesaminimum of0.5mMconcentrationintheentirepHrange ofthetitration.Forlesssoluble compounds, a good alternative tool is the UV/pH titration provided that the moleculehasapH‐dependentUVspectrum.Inthismethod,itisgenerallyenough if the compound dissolves in a concentration of 10‐500 μM depending on its molar absorptivity, ε. Both potentiometric and UV/pH titration methods are stronglysupportedcommercially,andtheavailableautomatedinstrumentssuch astheGLpKaanditsfollowuptheSiriusT3automatedanalyzers(SiriusUK)are widelyused.Intherecentyears,capillaryelectrophoresis(CE)hasprovedtobea verypowerfulpKadetermination method,being moresensitiveandless sample consuming[11,16].SomeothermethodssuchasNMR/pHtitration[55],CD/pH titration [56], and chromatographic technique [57] have also been applied for specialcases,butsofarhavenotbecomeroutinetechniques. 25 Chapter1 1.3.1.1. Potentiometricmethod Procedure.Inpotentiometrictitration,thepHofa1‐5mMsolutionofasampleis preciously measured with a carefully standardized combined glass electrode upon addition of small volumes of a strong acid (e.g. HCl) or base (e.g. KOH) volumetricsolution.Themeasurementisperformedinastirringsolution,under an inert gas atmosphere (argon or nitrogen) while the ionic strength of the solutioniskeptconstantusinganinorganicsalt(e.g.0.15MKCl),andthetitration cellisthermostatedusuallyat25.0±0.1°C.Typicalsamplevolumefortitrationis 5‐15ml,butmeasurementinaslessas1mlsolutionhasbeenreported[58].The concentration of the titrant is generally 0.5 M in order to avoid considerable dilution upon titration. The potentiometric titration can be used as a direct approach for pKa measurement, when the tested compound is a (relatively) strongacid/basetoproduceenoughpotentialchange(bigjump)inthetitration curve. Otherwise, the “Calvin‐Δml” difference‐titration is a useful and widely appliedmethod.Here,thepKavalueisobtainedfromthedifferencebetweenthe titration curve of a tested compound and a “blank” titration (see below). This approachisabuiltinfunctioninpKaanalyzers. Calculation. The pKa value can be calculated according to the HH equations (Equations1.1c‐1.4c).ThepHismeasuredandthetermlog([protonated]/[non‐ protonated]) is obtained from the mass balance of the titration data. In automatedanalyzersbuiltinprograms(e.g.,Refinement‐ProTM)calculatethepKa. First,thetitrationcurveisconvertedtotheBjerrumplot(theaveragenumberof boundprotons/molecule, n vs.pH),wherethepKavalueisequaltothepHat n = 0.5(foramultiproticcompound:secondpKaat n =1.5,thirdat n =2.5,etc.).The obtained raw values are then further refined by a nonlinear least squares method. The adjustable parameters are the concentration of the material, acid/baseerrorofpHmeasurement,carbondioxidecontent,etc. Accuracy. This method with the above experimental parameters allows the measurement of precise pKa values in a range from 2 to 12 with a standard deviation SD = ± 0.01‐0.03. By using a glass electrode of excellent quality, performing proper electrode calibration, excluding the presence of ambient carbondioxideasmuchaspossible,andaccuratelydispensingverysmalltitrant volumes (0.01 ml or even smaller) potentiometry in aqueous solution can be appliedtoaconcentrationaslowas0.1mM(accordingtosomeauthorsaslowas 0.01mM).Ofcourse,theaccuracyandreproducibilityoftitrationsinsuchdiluted solutionsismuchless(SD=±0.10‐0.15).Similarly,theprecisionofthemeasure‐ mentdecreasesoutofthepHrangeof2‐12. Calibration. Electrode calibration is a fundamental step in pH‐metric pKa determination.Astandardized“Four‐parameterprocedure”developedbyAvdeef et al. [59] is widely used. A known concentration of HCl is titrated with KOH (frompH1.8to12.2)understandardexperimentalconditions(seeabove).Data from this “blank” titration are used to convert the operational pH scale to the concentrationscale(pcH=−log[H+])byamulti‐parametricequation. 26 Physicochemicalprofilingindrugresearchanddevelopment pH=α+SpcH+jH[H+]+jOHKw/[H+] (1.32) The parameters are determined by a weighted nonlinear least‐squares procedure.Theinterceptparameterαinaqueoussolutionmainlycorrespondsto thenegativelogarithmoftheactivitycoefficientofH+attheworkingtemperature and ionic strength. The jH term corrects pH readings for the nonlinear pH response due to the liquid junction and asymmetric potentials in moderately acidicsolutions,whilethejOHtermcorrectsthehigh‐pHnonlineareffect.FactorS accounts for the fact that a particular electrode may not have 100% Nernstian‐ slope and Kw is the ionization constant of water. Typical aqueous values of the adjustableparametersat25°Cand0.15Mionicstrengthare:α=0.08±0.01,S= 1.001±0.001,jH=1.0±0.2,andjOH=−0.6±0.2. Advantages/drawbacks. Potentiometry is a simple, fast, and precise method for pKa determination. The smallest practical volume of sample solution is about 5ml.Thisrequires1.5mgofsampleforacompoundwithMw300toachievethe 1mM concentration which is ideal for titration. For reliable pKa, 2–3 parallel measurements are necessary, so the sample consumption reaches 3–4.5 mg. A titration between pH 2–12 typically takes 20‐40 min to perform. With an automated instrument (e.g. GLpKa) 30‐40 titrations could be performed during one 24‐h day [60]. So, the maximum capacity is about 10‐12 compounds/day. Thisisarelativelylowthroughput.Themainlimitationoftheapplicationofthis technique is the poor solubility of compounds. In such cases, the co‐solvent method can be applied (see Section 1.3.1.4). Further on, it is difficult to handle impureorunstablecompounds(e.g.,certainesters,diphenols,etc.). 1.3.1.2. UV/pHtitration Procedure. In spectrophotometric pKa determination method the change in the UVspectrumuponionizationisregistered.SuchapHdependentUV‐spectrumis obtained if the ionizable group is near to the chromophore of the molecule. In traditional UV/pH titration two aliquots of typically 10‐50 μM solutions of a samplearepreparedineither0.01(or0.001)MHClor0.01(or0.001)MNaOH, with the total ionic strength of 0.15 M. In one solution the molecule is fully ionized while in the other fully unionized. By mixing the two stock solutions underprecisepHcontrol,5‐6solutionsarepreparedinarathernarrowpHrange (± 0.6 unit) around the expected pKa. Their absorbance is measured at a wavelength where the difference in the absorbance between the ionized and unionized form is the largest. Recently, this time‐consuming process has been automated (GLpKa with a D‐PAS attachment). In a titration cell, the solution of thesampleistitratedacrossapHrangethatincludesthepKavalue(s)andmulti‐ wavelength UV spectra registered at each pH with the help of a fiber optics dip probeimmersedintothetitrationcell[60,61]. Calculation. In traditional UV/pH titration, the pKa value can be calculated from thepHofthesolutionandtheabsorptiondatameasuredatasinglewavelength using the HH Equations 1.1c‐1.4c. The pKa of a compound is obtained as an 27 Chapter1 average value calculated from the solution series. This method is applicable for thedeterminationofasinglepKa,ormultiplepKavaluesiftheyarewellseparated (>1.5 pH units). In the D‐PAS technique, target factor analysis (TFA) is used to deduce the pKa value(s) of a sample from an absorbance matrix [60]. This techniqueisabletohandlemultiproticmoleculeswithoverlappingprotonation. Accuracy.TheprecisionofpKadeterminationbytraditionalUV/pHtitrationdoes not reach that of pH‐metry, where the standard deviation can vary between ± 0.05‐0.10. However, according to a recent validation study, the D‐PAS tech‐ niquewithaSD=±0.02hassimilarprecisiontopotentiometry[62]. Advantages/drawbacks. The spectrophotometric method is usually more sensitive than potentiometry. The measurements can be performed at lower sampleconcentrationallowingthepKadeterminationoflesssolublecompounds directly in aqueous medium, while for water‐insoluble materials the co‐solvent methodcanbeeasilyapplied.TheD‐PASisafasttechnique,onetitrationtakesup 30 min and is sample conserving, usually 1‐2 mg of sample is enough for 3 parallel measurements. One limitation of spectrophotometry is that if the distance between the ionization and the chromophore center is greater than three sigma bonds then the pH‐dependent spectral shift will be too small for measurement. Another limitation is if the absorption maxima of the compound occurs at a low wavelength (< 230 nm) then background noise disruption increases considerably. Traditional UV/pH titration is a very slow, time‐ consuming process, while the capacity of the D‐PAS technique is similar to potentiometry(10‐12compounds/day).UV/pHtitrationwasusedfordetermina‐ tionofmicroconstantsinseveralcases(e.g.repaglinide[63],moxifloxacin[64]) when the shift in the UV spectrum is due to the ionization of a given functional group. 1.3.1.3. Othermethods NMR/pHtitration.NMR/pHtitrationcanalsobeusedforpKameasurementbased onthefactthatthechemicalshiftofNMR‐activenucleiisgoverned(amongother factors)bytheprotonationstateofionizablegroups.Sinceprotonationdecreases the local electron density, a selected nucleus in the vicinity of the ionizable site exhibitsadifferentshiftintheionizedandunionizedstates.Aplotδobsvs.pHhas asigmoidalshapewithaninflectionpointatpH=pKa. Generally, NMR/pH titrations have been carried out in aqueous solutions using D2Oasasolvent.AlthoughglasselectrodesoperateproperlyinD2O,acorrection factor of 0.40 has to be added to the measured pH to get the true pD value. To avoidthiscorrection,NMR/pHtitrationmaybeconductedinasolventmixtureof H2O/D2O(90/10v/v)andthewaterpeakhastobesuppressedbyanappropriate method. Frequently, the whole titration is performed in a single NMR‐tube and the pH is measured with a long, thin glass electrode. This method has been extended for the measurement of low pKa values (between 0 and 2), where po‐ tentiometry is no longer applicable. Since at such low pH a glass electrode has 28 Physicochemicalprofilingindrugresearchanddevelopment significantacidityerror,dichloroaceticacidwasproposedasanNMR“indicator molecule”forinsitumonitoringofthepHinstrongacidicsolutions[55].ThepKa valuesofindividualgroupsoflargebiopolymershavebeenreportedasmeasured byNMRtechnique[65]. The main advantage of this technique compared to potentiometry is the capa‐ bility of selective monitoring of ionization of a given functional group in multi‐ protic molecules with overlapping protonation. Thus, this methodology has become the chief approach of microspeciation as reviewed recently [34]. The acid/base profiling of imatinib [66] and cetirizine [33], measured by NMR/pH titrationwasreported. Capillary Electrophoresis (CE). The application of CE for pKa determination has been intensively growing in the past decade as reviewed [7,16,67,68]. The method utilizes the change in electrophoretic mobility of a compound with changeinpH.Theeffectivemobility(μeff)ismeasuredatvariouspHvaluesand pKaisobtainedfromtheplotofμeffvs.pH.Theexperimentalconditionseffectthe pKa determination such as buffer type and ionic strength, applied voltage, detectionmethod,etc.arediscussedasdetailedbyHenchozetal.[16]. Inthistechnique,thesampleconsumptionissmall(ng),andimpuresamplescan be handled due to the separation upon the analysis. It is rather universal, since different detection methods can be coupled to CE [69]. The precision is good enoughandagreementwithothermethodsisacceptable,about±0.2pKaunitsin arangefrom2to10,butcanbemuchweaker(±0.5)outofthispKarange.The method is sensitive for several factors, among them temperature which is cardinal. Today, CE is a good tool for high throughput pKa measurement. The instru‐ mentationisfullyautomatedusingamultiplex96‐channelCEwithUVdetection (CombiSep,Ames,USA)andmorethan150samples/daycanbemeasured[70]. Spectral Gradient Analysis (SGA). To further increase the throughput of physicochemicalprofiling,arapidpKadeterminationmethodwasdevelopedand reported first as “pH‐gradient titration” [71]. Later, after the launch of a com‐ mercialinstrument(ProfilerSGA,Sirius)itisreferredtointheliteratureasthe SGA method. In this technique, a pH gradient flow – very linear in time – is created by mixing appropriate acidic and basic buffers. The sample is injected into this pH gradient flow which passes through a diode array UV spectro‐ photometerandthespectraareregistered.ThepHisnotmeasuredbutestimated fromthetimeelapsedsincethestartofthegradientgeneration.ThepKavalues aredeterminedfromchangesinabsorptionasafunctionofpH.Thecalculationis basedon eitherthefirst derivativeplotoftheabsorptionspectrumforsamples withonlyasinglepKa(orwellseparatedpKavalues)orontheTFAapproachfor compounds with weak spectral change or overlapping ionization [60]. The precisionofthemethodisevidentlylowerthanthatofothermethods,butresults of a comprehensive validation study show good agreement with literature data [72]. The SGA method allows pKa measurement within 4 min leading to high 29 Chapter1 throughput capacity. The present available automated instrument (Sirius T3) containinganautoloadermodule(roboticarm)utilizesfour48‐positionvialtrays forsamples.Itenablesthemeasurementof240compounds/day.Lowsolubility andlowmolarabsorptivitymaybelimitationsoftheSGAmethod. 1.3.1.4. Co‐solventmethod DeterminationofpKausingtheabovediscussedmethodsisoftenhinderedbythe lowwatersolubilityofthesamples.Itisafrequentproblemtodaysincethenew moleculesindrugresearcharelesswater‐solubleandmorelipophilic.Forwater insoluble compounds, the co‐solvent method can be used. In this approach, the apparent ionization constants, psKa values, are measured in different ratios of organic solvent/water mixtures. The aqueous pKa value is obtained by extra‐ polationtozeroorganiccontent.Theco‐solventmethodisprimarilyusedinpH‐ metry,butitcanbeappliedinUV‐spectroscopyandCEtechniquesaswell. Manywatermiscibleorganicsolventshavebeenusedsuchasmethanol(MeOH), ethanol (EtOH), propanol, DMSO, dimehtylformamide (DMF), acetone, and tetrahydrofurane(THF).Sincemostliteraturedatahavebeenaccumulatedfora MeOH/water solvent mixture and it is generally accepted that MeOH shows a solvationeffectclosesttowater,MeOHisnormallychosenasanorganicsolvent ofchoice[11,16,68]. Different extrapolation methods are known, but the Yasuda‐Shedlovky (YS) extrapolation has proven to be the most reliable. Here, a linear correlation is establishedinaplotofpsKa+log[H2O]vs.a/ε+b,wherelog[H2O]isthemolar water concentration of the given solvent mixture, ε is the dielectric constant of the mixture, and a and b are the slope and intercept, respectively. The aqueous pKavaluescanbeobtainedforlog55.5and1/78.3,themolarconcentrationand dielectric constant of pure water, correspondingly. The dielectric constant of MeOH/watermixturesislowerthanthatofwaterandtheextentofionizationis suppressed,thus pKa values of acids are shifted higher while those of bases are toward lower values. The slope of the YS relationship is positive for acids and negative for bases. The YS procedure offers many benefits over the traditional plotofpsKavs.Rw(wt%oforganicsolvent)whichoftenshowsa“hockey‐stick”or “bow”shape,sometimesatRw>60wt%anS‐shapecurve.Properelectrodecali‐ brationusingfourparameterproceduresinthesolventmixtureiscrucial[73]. Accordingtoacomprehensivevalidationstudy,thereproducibilityandprecision of the method, based on 431 separate titrations in the interval of 15‐65 wt% MeOHcontentusing25modelcompounds,wasfoundtobegood(SD=±0.05). Extrapolationfromamethanol‐richregion(Rw:40‐60wt%)givesanerrorinpKa notgreaterthan±0.2forweakacidsand±0.1forweakbases[74]. Sincenotallcompoundsdissolveinasingleorganicsolvent(e.g.methanol),anew multicomponent co‐solvent system significantly improving the solubility of pharmaceuticalcompoundswasrecentlydevelopedforpKadetermination[75,76]. The mixture consists of an equal volume of MeOH, dioxane, and acetonitrile 30 Physicochemicalprofilingindrugresearchanddevelopment (referredtoasMDM)dilutedinwatertoobtaintherequiredco‐solventsystem. This system enables pKa measurements by potentiometry (and also by UV/pH titration) for a wide range of poorly soluble compounds. Since solubility considerablyincreasesintheMDMsystem,measurementscanbeperformedina lowerproportionoforganicsolvent,thusthelong‐distanceextrapolationcanbe avoided.ThelinearityoftheYSrelationshipisvalidupto55wt%MDMcontent. Validation based on 50 compounds showed good reproducibility (SD=±0.01‐0.08)andtheagreementofpKavaluesextrapolatedbythismethod withvaluesmeasuredbyothermethodsisverygood(<0.10unit). The SGA method has been extended with measurements in 20 wt % MDM content, and general calibration equations were set up for acids and bases (pKa(aqueous)=apsKa (20%MDM)+b),soasinglepointestimationmayproviderapid aqueous pKa values for water‐insoluble compounds in the early phase of drug research[76]. 1.3.1.5. Decisiontreeformethodselection The selection of a suitable method must be based on the properties of the compoundtested.Figure1.12showsasimpledecisiontreeformethodselection usedinthelaboratoryoftheauthor[4]. compound pH-metry YES UV/pH titration 0.5 mM solubility in water NO pH sensitive UV spectrum Co-solvent method pH-metry YES 1-5 µM solubility in water NO 0.5 mM solubility in MDM/water YES NO Single point estimation 80% DMSO pH-metry YES YES 0.5 mM solubility in 80% DMSO NO 1-5 µM solubility in MDM/water NO YES Co-solvent method UV/pH titration NO pKa prediction Figure1.12.DecisiontreeformethodselectionofpKameasurement 31 Chapter1 Table1.4.MethodsforpKadetermination Sample Method potentiometry UV/pH titration solu‐ amount, bility, mg mM 3‐5 Throughput high purity speed1 capa‐ Precision city2 >0.5 necessary 20‐30 10‐12 Instrumen‐ tation high GLpKa,SiriusT3 (Sirius,UK) traditional 1‐2 >0.01 necessary 6‐8 hours 1 medium pH‐meter+ spectrophotometer automated 1‐2 >0.01 necessary 30 10‐12 high GLpKa+D‐PAS, SiriusT3(Sirius,UK) NMR/pH titration 1‐2 >0.5 2‐3 high NMRspectrometer singlechannel <<1 >0.01 not necessary 30 20 medium CE multiplexed <<1 >0.01 not necessary 30 150 acceptable CePro9600 (CombiSep) >0.01 necessary 4 240 acceptable Profiler‐SGA, SiriusT3(Sirius,UK) CE* SGA† 1 *CapillaryElectrophoresis †SpectralGradientAnalysis not 2‐3 necessary hours 1 min/comp. sample/day 2 1.3.2. logSdetermination Severalmethodshavebeendevelopedforthemeasurementofbothequilibrium and kinetic solubility including traditional and high throughput techniques. Ex‐ cellent reviews [7,16,39,41] have surveyed the state‐of‐the‐art techniques. Below,afterashortsummaryofkineticsolubilitymethods,approachesforequili‐ brium solubility measurement are discussed focusing on good laboratory practice(GLP). 1.3.2.1. Methodsfordeterminationofkineticsolubility Concerning the large number but small content, samples in the early phase of drug discovery are subjected to compound‐saving and HT methods which are suitable for the measurement of kinetic solubility. In the turbidimetric method introduced by Lipinski et al. [77] small aliquots (0.5 μl) of DMSO stock solution areaddedat1min.intervalstoaqueousbuffers(originally,2.5mlofpH7phos‐ phate buffer) until the compound precipitates from the solution reaching the maximal (but not yet the equilibrium) solubility. The turbidity caused by the precipitationismeasuredbylightscatteringinthe620‐820nmrangewithaUV detector. In nephelometric [78], direct UV [79] and ultrafiltration‐LC/MS [80] methodstheaboveprincipleisadaptedto96‐wellplateusingdifferentdetectors (nephelometer,diodearrayUVand MS,respectively).Inthe twolater methods, 32 Physicochemicalprofilingindrugresearchanddevelopment the precipitate is separated from the solution by filtration (or centrifugation) beforetheconcentrationmeasurement.Theultrafiltration‐LC/MStechniquehas theadvantageofhighsensitivityandthecapabilityofhandlingimpuresamples. Commercially available instruments (Nephelostar, BMG; Nepheskan Ascent, Thermo Labsystem, μSOL, pION) use fully automated liquid dispensing systems and provide high capacity (measurement of 200‐300 compounds/day). The presence of DMSO in the kinetic solubility experiments may considerably affect theresultsinahighlycompound‐dependentway,thusitispracticaltokeepthe DMSOataminimumlevel(lessthan0.5%). Themaindisadvantagesofkineticsolubilitymeasurementsarethelackofstan‐ dardization,poorreproducibility,anddifficultiesinthecomparabilityofresults. 1.3.2.2. Methodsfordeterminationofequilibriumsolubility 1.3.2.2.1. Saturationshake‐flaskmethod(SSF) The SSF method is the standard approach for the determination of equilibrium solubility which when properly performed provides high quality data. It is a simple but very time‐consuming procedure and requires lots of manual work. The solution of the tested compound containing excess solid is prepared in aqueous buffer using a small (2‐5 ml) glass vial. The heterogeneous system is cappedand vigorouslystirredatachosentemperature(usually 25°Cor 37°C) foraspecifiedtime(24,48horlonger)untiltheequilibriumhasbeenreached. After that, the two phases (solid and liquid) are separated by sedimentation, centrifugation, or filtration. Upon diluting sample aliquots with the solvent, if necessary, the concentration of the saturated solution is measured by an appropriate method, most frequently by UV spectroscopy or HPLC. Despite the longevityofSSFuse,thereinnoacceptedstandardwaytocarryoutthismethod. Published solubility studies show great differences in the experimental conditionsused,particularlyconcerningthetimeofequilibration,themethodof phaseseparation,andthecontrolofpHduringthemeasurement[37,38,41]. Recently, in a comprehensive study published by Baka et al. [81] the most important experimental factors influencing the measured equilibrium solubility bytheSSFwereinvestigated(seesomeresultsinSection1.4.2)andastandardized protocolwasproposedforGLP[82].Thefollowingconditionsaresuggested: themeasurementmustbecarriedoutatcontrolledtemperaturewithprecision±0.1°C, SörensenphosphatebuffercanbeusedbetweenpH3‐7,whileBritton‐Robinsonbuffer canbeusedinawiderpHrangefrom2.5to11.5.HClofappropriateconcentrationcan beusedbelowpH2.5, thepHofthesolutionmustbecarefullycontrolledduringthemeasurement,advisably beforeandaftertheequilibration, to avoid the difficulties in sampling, only a small (~ 5‐10 mg/5 ml) excess of solid shouldbepresent, aminimumof24hisnecessarytoreachtheequilibrium,thistimeshouldconsistof6h ofstirringand18hofsedimentation,butincaseofverysparinglysolublecompounds longer stirring time may be necessary for equilibrium, so in the most rigorous 33 Chapter1 application of SSF, the required time of equilibration must be determined from compoundtocompound, the safest technique of phase separation is sedimentation which assures a hetero‐ geneous system until equilibrium has been achieved; if an opalescent solution is formed then the phase separation can be done by centrifugation while the most erroneousfiltrationshouldbeavoided(seeresultsinSection1.4.2below), acompoundexistinginameta‐stablepolymorphformcanbetransformedintoamore stableoneduringthedurationofsolubilitymeasurement,thustheanalysisofthesolid phase(byX‐raypowderdiffractionorthermo‐gravimetricmethods)attheendofthe experimentishighlyrecommended. Using the above listed conditions the equilibrium solubility of more than 50 com‐ poundswasdeterminedwithastandarddeviationoflessthan4%inourlaboratory. 1.3.2.2.2. Potentiometricmethods Theprincipleofthepotentiometricmethodsisbasedonthatcharacteristicshift ofthetitrationcurvecausedbytheprecipitationoftheunionizedformofacom‐ pound from a solution. Potentiometric titration was introduced for equilibrium solubility measurement by Avdeef et al. [83‐85]. The dissolution template tit‐ ration(DTT)methodusespKaandlogPvaluesasinputparameters.LogPisused to estimate the intrinsic solubility based on a Hansch‐Yalkowsky type equation (log So = 1.17 – 1.38 log P). Using the pKa and the estimated intrinsic solubility, the DTT procedure simulates the entire titration curve before the assay begins. Thiscurveservesasatitrationtemplate(theoptimalquantityofthetestedcom‐ poundforthetitrationissuggestedbythesimulation)andalsoasaguideforthe righttitrationprotocol(howtheinstrumentdispensesthetitrantandcollectsthe pHdata)inthecourseofthetitration.ThetitrationstartsatpHvalues,wherethe compoundisunionizedandformsasuspension(solidmaterialispresentinthe solution).Thetitrantisdispensedaccuratelyandslowlyintotheslurry,todrive thepHofthesolutioninthedirectionofdissolution.Typically,a3‐10h(some‐ timeslonger)timeframeisrequiredfortheentireequilibriumsolubilitydatacol‐ lection(20‐50pHpoints)[41].Themethod,whenperformedwiththepSOLtit‐ rator(pION,US),providesaprecisesolubility‐pHprofilewithoutassumingaHH relationship and is much faster than the SSF method but still has a very low throughput. The novel potentiometric procedure (CheqSol) has been developed recently for rapid measurement of solubility using the instrument called the GLpKa‐D‐PAS (Sirius, UK). In this method, the equilibrium solubility is actively sought by changing the concentration of the neutral (unionized) form of a compound by addingacidorbasetitrantsandmonitoringtherateofthechangeofpH,dueto precipitation or dissolution in a process called “Chasing Equilibrium”. In this method,thetitrationisstartedatpHvalue,wherethecompoundisfullyionized and dissolved and performed toward the direction of pH where the unionized form precipitates. The turbidity of the solution caused by the precipitation is detectedwithafiberopticdipprobe.Withthismethodboththekineticsolubility andtheequilibriumsolubilitycanbedetermined.Thekinetic solubilityvalueis 34 Physicochemicalprofilingindrugresearchanddevelopment obtained from the concentration when the first precipitation of the unionized form appears in the solution, while the equilibrium solubility is obtained from actively seeking the equilibrium pH where an equal amount of the sample is precipitating and dissolving per unit of time [86]. The CheqSol method is faster (typically 30–60 min/compound) because the intrinsic solubility is determined instead of the entire solubility‐pH profile. Then HH equation is used for the calculationoftheapproximatelogS/pHprofile.ItwasvalidatedagainsttheSSF methodandexcellentagreementofsolubilityresultswasfound[87]. 1.3.2.2.3. μDISSmethod A miniaturized rotating disk dissolution instrument, called μDISS ProfilerPLUS (pION,US)hasbeendevelopedforcharacterizingtheintrinsicdissolutionratein early preformulation. This apparatus is also suitable for the measurement of equilibriumsolubilityofsparinglysolublecompounds,providedenoughmaterial isusedtomaintainthesaturation[41,88,89].Inthisprocedure,5mgofdrugare compressed into pellets and inserted into a rotating disk carrier containing an embedded magnetic stir bar at its bottom. This assembly is placed into a glass vialfilledwithasmallvolume(1‐3ml)ofaqueousbufferasthedissolutionmedi‐ um.TheconcentrationismeasuredwitharapidinsitufiberopticUV(diodear‐ ray) detector. The instrument employs six parallel dissolution vessels and eight channels of UV detectors which provide better capacity above the SSF method. Overthehighprecision,furtheradvantagesofthismethodare:(i)anypolymorph changesduringdissolutioncanberecognizedand(ii)thelongerincubationtime neededtoestablishthetrueequilibriumofthemoststableformofasolidmaybe evidentinthedissolutioncurve[39]. 1.3.2.2.4. Highthroughputmethods SomemethodssuitableformediumorhighthroughputdeterminationoflogSwere also described. The miniaturised shake‐flask (MSF) method developed by Glomme etal. [90,91] is a compound saving and fast method, thus it is frequently used in pharmaceutical companies. Typically, 0.1‐0.2 mg solid powder is introduced to a speciallydesignedfilterchamberandasmall(e.g.2ml)volumeofaqueousbuffer isadded. Purpose‐built filtercaps arefirmly attached andthe vials areshaken at constant temperature for 24 h. The filter‐containing cap compartments are then depressedtoeffectseparationofthesolidandthetopcompartmentsolutionsare analyzed by fast gradient RP‐HPLC. The throughput is just medium, as 20 compounds/week can be measured. The MSF method was further developed for HTmeasurementsbyZhouetal.[92]wherea96‐wellplateisusedasthesourceof thesamplesandDMSOstockswereevaporatedviaaGeneVacevaporator. Those96‐wellplatebasedHTmethods(originallydevelopedforkineticsolubility measurement),wheretheincubationtimeislongenough(e.g.μSOLmethod,[39]) and the effect of DMSO content is eliminated, are also suitable for equilibrium solubility determination. Generally, in these modified‐microplate methods the 24h incubation time is adequate to reach the solubility equilibrium [39]. 35 Chapter1 However,duetosmallvolumes,theprecisepHcontrolduringthemeasurement may be problematic. In the lyophilized solubility assay (LYSA) the sample is dispensed into a microtiter plate along with 10 mM DMSO solution then the organicsolventisremovedbylyophilizationandaqueousbufferisadded.During a 24 h incubation period the plate is agitated by a shaking mechanism, then filtratedandtheconcentrationismeasuredusingaUVplatereader[93].Another promising HT procedure is the PASS (Partially Automated Solubility Screening) method,wherethecompoundsaresuspendedinheptaneanddispensedintothe platewells,thenheptaneisevaporatedbeforebufferisadded[94]. 1.3.2.3. Specialapplications Themethodsdescribedabovehavebeenappliedforspecialpurposes.Potentio‐ metric titration, according to the CheqSol approach, has been reported to study the solubility of polymorphs. A new method named “potentiometric cycling for polymorph creation” (PC)2, was developed to generate the most stable poly‐ morph in aqueous solution [95]. It was applied to sulindac producing two polymorphs including a new, more stable one. It was found that their intrinsic solubilitydifferbyafactorofseven,whichismuchlargerthanthatofanyearlier measureddifferencebetweenpolymorphs. Table1.5.MethodsforlogSdetermination Method Detection LOD, μg/ml Throughput speed, capacity, Precision min/comp. samp./day Instrumen‐ tation forkineticsolubility turbidimetric UV nephelometric lasernephe‐ lometer 5 15 5 4 direct‐UV UV 2·10‐3 ultafiltration‐ LC/MS MS 0.1 50 low 300 low Nephelostar, Nepheloscan 4 300 medium μSOL 6 200 medium LC/MS forequilibriumsolubility SSF UV;HPLC Potentiometric DTT pH‐metry 36hours <1 high ‐3 5·10 3‐10hours 1‐5 high pSOL CheqSol pH‐metry 0.1 30‐60 10‐15 high GLpKa;SiriusT3 UV 1 24hours 6 high μDISS ProfilerPLUS MSF UV;LC/MS 1 3‐100 medium μSOL UV 0.1 24hours 18‐24 hours 100 acceptable μSOL μDISS modified‐plate HT 1 LYSA UV 1 24hours 100 acceptable PASS UPLC 103 24hours 100 acceptable 36 Physicochemicalprofilingindrugresearchanddevelopment Thebiorelevantsolubilityvaluesaremoreandmorerequiredindrugdiscovery and development (DD&D). An optimized 96‐well HT UV solubility method was adaptedtomeasuresolubilityofdrugsinbiorelevantmediasuchasFaSSIFand FeSSIF solutions [96]. The method provides reliable data using a very small amount of sample and small volumes of the expensive FaSSIF/FeSSIF compo‐ nents.TheμDISSmethodwasalsofoundusefulformeasurementinbiorelevant media and temperature [97]. The study has revealed that the majority of the testeddrugsexhibitedhighersolubilityinthesemediathaninpurebuffers. 1.3.3. logPdetermination Since log P is the oldest parameter in physicochemical profiling, several well‐ established experimental methods are available for its determination. Vast amountsofliteraturehavedescribedthetheoryandpracticeoftheusedmethods [e.g.11,46,98‐101].AspectsfromtheGLPguideforlogPmeasurementshavealso been published [98,101,102]. The recent reviews provide a comprehensive surveyaboutthelatestdevelopmentsinHTtechniques[6,9,14‐16]. TwotypesofmethodscanbedistinguishedforlogPdetermination:(i)thedirect approaches, where log P is directly obtained from the measured data (shake‐ ‐flask, stir‐flask, filter chamber, dual‐phase potentiometric, etc.) and (ii) indirect (chromatographic, CE) techniques, where the measured parameter has a linear relationshipwithlogPandlogPiscalculatedusingcalibrationequations.Inthis chapter,thedirectmethodsareoverviewed,outoftheindirectmethodsonlyTLC ispresented,whileotherslikeHPLC,MECK,etc.usedforlogPmeasurementare discussedelsewhereinthebook. Inordertofacilitatethecomparisonoftheircapacity,Table1.6summarizesboth typesofmethods. 1.3.3.1. Shake‐flask(SF)method The traditional SF method is the reference and most widely used approach of logPdetermination. Procedure.Inadvance,thetwophases(n‐octanolorotherusefulpartitionorganic solvent that is immiscible with water and aqueous buffer) must be mutually saturated with vigorous agitation then filtered or centrifuged. The tested substanceisdissolvedintheaqueousphaseandintroducedintoanappropriate glassvial.Octanol(orotherorganic solvent)isaddedinarequiredvolumeand the system is shaken at a constant temperature for a period long enough for equilibrium to be achieved (generally 1 h). After separation of the phases by centrifugation, the concentration is measured using an appropriate method, mostlyUVspectroscopy.Concerningthedifficultiesofthepreciseanalyticalwork with octanol, it is a common practice to measure the concentration decrease in theaqueousphasebydetectingtheabsorbancebeforeandafterthepartition. Accuracy, sources of the experimental error. The SF method is suitable for logP measurementintherangefrom–2to5havingaSD<0.05,providedthatoptimal 37 Chapter1 experimentalconditionsaremaintained.Manyfactorscanaffectthereliabilityof the measured log P values increasing the experimental error. One of them is undoubtedly the applied extreme phase ratio necessary in the case of lipophilic compounds(logP>3).Accordingtoourexperiences,thehighestphaseratiothat can be used without a considerable increase in error is R = 500 (e.g. 50ml aqueousbuffer:0.1mloctanol).However,intheoppositecasewithhydrophilic compounds, when more octanol has to be used, the sampling from the lower aqueous phase may be problematic, thus it is advisable to remove the upper octanollayerbeforealiquotsaretaken.Glassandsurfaceadsorption,formation of stable emulsions, and the presence of impurities in the sample have often influencedtheresults. Advantages/drawbacks. The main advantage of the SF method is its simplicity, sufficient accuracy, and applicability to non‐ionizable compounds. But it has some well‐known shortcomings, such as being tedious and time‐consuming, difficultieswithmaintainingaconstanttemperatureduringthewholeprocedure, requiringrelativelyhighamountsofsampleandsolvent.TheSFmethodcannot be used for UV inactive compounds unless alternative detection methods are employed,andsoon. 1.3.3.2. Potentiometricmethod Dualphasepotentiometrictitrationusingautomatedinstrumentshasbecomethe “goldstandard”oflogPdetermination(forionizablecompounds)inthepastten years [11,60,100]. It consists of two titrations of the tested compound. One is performedwithoutthepartitionsolventandprovidestheaqueouspKavalue.The second is done using the same conditions but in the presence of a partition solvent(e.g.octanol)withintensivestirringupontitrantaddition,whilestopping it when the pH is measured. If the unionized form of the compound partitions intooctanolthenthetwotitrationcurveswillbedifferent,duetoashift(similar to what was discussed in the co‐solvent pKa method). From the dual‐phase titration, the apparent poKa value is obtained. Log P is calculated from the differences in pKa values and the phase ratio. A large shift indicates high lipophilicity(seealsoinSection1.4.3). This method allows the log P determination in a range from ‐2 to 6, with very high precision (SD = ± 0.01). The agreement with the SF method is excellent according to validation studies [103,104]. However, it has limited capacity and cannot be used for compounds with pKa out of the established measurable pH range.Afurtherdrawbackisthatonlyalimitedphaseratiocanbeapplied(inour practicewithGLpKa:20mlwater:0.05mloctanolforlipophiliccompoundsand 5mlofwater:15mlofoctanolforhydrophiliccompounds).Anewlydeveloped instrument(SiriusT3)hasfurtherincreasedefficacyandmeasurementispossible inaslowas1mlaqueousphase. 38 Physicochemicalprofilingindrugresearchanddevelopment 1.3.3.3. IndirectlogPmeasurementmethods Because of the drawbacks and limitations of direct methods, numerous alternative procedures have been developed and applied. Several micellar, microemulsion, vesicle electrokinetic chromatographic systems, and reversed‐ phase chromatographic methods (RP‐TLC, RP‐HPLC) can be used to estimate lipophilicity. Some excellent reviews on the use of separation methods for indirectlogPdeterminationhavebeenpublished[e.g.105‐106]. AlthoughRP‐HPLCismorewidelyusedtechniqueforlogPestimation[105],RP‐ TLC undoubtedly has some unique advantages, including use of less expensive laboratoryequipmentandbeingeasytoperform.Simultaneousrunningof15‐20 compounds on one plate can significantly reduce the analysis time per com‐ pound. The method is based on the linear relationship between logP measured bytheSFmethodandthelogarithmofchromatographicretentionexpressedas the RM value. RP‐TLC has been successfully applied for logP measurement of highlylipophilicmoleculesusingcalibrationequationsobtainedwithstructurally related compounds [e.g. 107‐109]. Recently, a validated RP‐TLC method was proposedforparallelestimationofthelipophilicityofchemicallydiverseneutral compoundsorweakacidsandbases[110].Tocoverawiderangeoflipophilicity, two optimized RP‐TLC systems were used: one for moderate lipophilic com‐ pounds(logP=0‐3)andanotherforhighlylipophilicmolecules(logP=3‐6). Two chemically diverse sets of compounds were selected to set up the general calibration equations. The method was tested with 20 randomly selected drugs and good agreement with SF results were found (SD < 0.15). With automated sampling and imaging detection of the compounds the method can be regarded as a possible alternative for rapid and acceptable accurate estimation of lipo‐ philicityofdrugcandidatesintheearlyphaseofDD&D. 1.3.3.4. Highthroughputmethods Attempts have been done to miniaturize the traditional SF method into a microtiter plate platform with robotic liquid handling and HPLC/UV [111] or LC/MS [112] detection. In these techniques the partitioning process is transferred to 96‐deep well plates and after equilibration, the detector signal produced by a sample from the octanol phase is divided by the signal from the aqueousphase.InareviewbyKerns[7],thecriticalelementsofthesemethods arediscussed.AcommerciallyavailableautomatedplatebasedinstrumentforHT logPdeterminationiscalledtheAlogP(Analiza,US). Regarding indirect (HPLC and MECK) log P methods, additional successful strategies were applied to increase the throughput and speed up the determi‐ nation time [16]. For example, the use of short columns and a high flow rate in HPLC,usageofUPLC,andmultiplexedMECKhavebeenreported.Thesemethods aresurveyedintheChapter2ofthisbook. 39 Chapter1 1.3.3.5. Decisiontreeformethodselection Themethodselectionstrategyfollowedinthelaboratoryoftheauthorisshown inFigure1.13. compound pH-metric YES ionizable YES solubility >1mM NO NO YES chromophore expected log P -2 5 YES Shake-flask NO NO RP-TLC RP-HPLC log P prediction Figure1.13.DecisiontreeformethodselectionoflogPmeasurement Table1.6.MethodsforlogPdetermination Method logP range Throughput sample amount, speed, capacity, Precision Instrumentation mg min/comp. samp./day direct methods shake-flask traditional automated (96-well plate platform) potentiometric -2 ↔ 5 2-10 180-360 2 high -2 ↔ 5 1-5 10 100 acceptable AlogP (Analiza Inc.) -2 ↔ 6 1-5 60 20 high GLpKa, SiriusT3 indirect methods 40 RP-TLC 0↔6 1-3 120 50 medium RP-HPLC -1 ↔ 6 0.01 15 100 acceptable MEEKC -1 ↔ 7 << 1 15 150 acceptable CePro 9600, MCE 2000 Physicochemicalprofilingindrugresearchanddevelopment 1.4. CASESTUDIES Inthischapterwepresentsomeusefulexamplesofphysicochemicalprofilingfor the illustration of methods discussed above. Mainly, such problematic compo‐ undshavebeenselectedwheretheroutinemeasurementisdifficultorhindered bycertainreasons.ThemostfrequentdifficultiesinthepKa,logS,andlogPde‐ terminationarethelowsolubility,instability,lackofUVactivityandpolymorph transition of the compound. The case studies introduced below can provide a possibletemplateforthemeasurementof“difficulttomeasure”molecules. 1.4.1. pKadetermination Case1. Sample: Method: RG‐1503 co‐solventmethod;potentiometrictitrationin methanol/watersystem Instrumentation:GLpKa(Sirius,UK) Thecompoundisamultiproticmoleculecontainingfourionizablegroups(Figure 1.14a:A‐D).ThetwopiperazineNatomsandthepyridineNareprotonaccepting basic centers, while the arylsulfonamide moiety represents a proton releasing acidicgroup.Theaqueoussolubilityofthecompoundislessthan0.5mMandit has no useful pH‐dependent UV spectrum in the pH range 3‐10, thus neither potentiometrynorUV/pHtitrationinanaqueousmediumcanbeused. Figure1.14.Co‐solventpH‐metryforpKadetermination: (a)structureofthesample(RG‐1503),(b)titrationcurvesindifferent methanol/watermixtures,(c)Bjerrumplots,(d)YSextrapolationcurves 41 Chapter1 The pKa values were measured by potentiometry using the co‐solvent method (seeSection1.3.1.4).Sixtitrationswereperformedinmethanol/watermixtures (40‐60 wt%) between pH 1.5 ‐ 12.5 in ~ 1 mM concentration of the sample, at 0.15 M (KCl) ionic strength, at 25.0 ± 0.1 °C temperature, under N2 atmosphere (Figure 1.14.b). From the obtained psKa values, the aqueous pKa values were calculatedbyYSextrapolation(Figure1.14d). Results:pKa1=2.02±0.22(Bgroup);pKa2=3.03±0.09(Cgroup); pKa3=7.35±0.03(Agroup);pKa4=11.40±0.09(Dgroup). Case2. Sample: nitrofurantoin Method: co‐solventmethod;UV/pHtitrationinMDM/watersystem Instrumentation: GLpKa+D‐PAS(Sirius,UK) Nitrofurantoinisawater‐insolublecompoundwhichhasoneacidicgroupandex‐ hibits a pH‐dependent UV spectrum. The pKa value was measured in an MDM/watersystembecauseitssolubilityishighenoughinthissolventmixture for the spectroscopic determination. A stock solution was prepared in 10 mM concentrationwithMDM,50μlofthisstocksolutionwasusedforthetitrationin 15mlof20‐50wt%MDM/watermixturesbetweenpH3‐10,at0.15M(KCl)ionic strength,at25.0±0.1°Ctemperature,underN2atmosphere.Figure1.15.cshows thepsKavaluesusedforYSextrapolation.TheextrapolatedaqueouspKa valueis: 6.87 ± 0.01 (R2= 0.9958), which is in good agreement with literature data measuredbyothermethods[76]. (a) nitrofurantoin (b) O N N 0.8 0,8 O 0.6 0,6 O Absorbance O 2N 1.0 1,0 H N 0.4 0,4 Absorbance 0.2 0,2 0.0 0,0 250 (c) 300 350 400 450 500 Wavelength (nm) (d) 17.6 25.4 36.6 43.4 48.8 9.1 psKa± SD 7.05 7.16 7.33 7.46 7.58 ± 0.05 ± 0.05 ± 0.05 ± 0.05 ± 0.05 9.0 psKa + log[H2O] t%) R(w psKa + log[H2O] = 60.4/ε 60.4/? + 7.837 8.9 8.8 8.7 8.6 13 14 15 16 1/ x 1000 Figure1.15.Co‐solventUV/pHtitrationforpKadetermination: (a)structureofthesample(nitrofurantoin),(b)pH‐dependentUVspectra, (c)apparentpKavaluesindifferentMDM/watermixtures,(d)YSextrapolation 42 Physicochemicalprofilingindrugresearchanddevelopment Case3. Sample: Method: lisinopril potentiometrictitrationinaqueoussolutionandin methanol/watersystem;NMR/pHtitration Instrumentation: GLpKa+D‐PAS(Sirius,UK);VarianInova600MHz spectrometer(PaloAlto,CA) Lisinoprilisatetraproticcompoundhavingtwoacidic(carboxyl)andtwobasic (a primary and a secondary amine) groups. Figure 1.16a shows the ionization processesofthemolecule.Thedissociationofthetwocarboxylgroupsishighly overlapping.Thesolubilityoflisinopril(0.22M)allowsthedeterminationofpKa valuesbythestandardpotentiometricmethodinaqueousmedium.However,the first dissociation constant falls into the low pH range which may cause uncertaintyofthemeasurement. For the characterization of the acid/base property of the molecule, three independentmethodswereapplied:potentiometryinaqueoussolutionandina methanol/watersystem,aswellasNMR/pHtitration.Inaqueousmedium,three titrationswerecarriedoutina2mMconcentrationsolution,betweenpH1.8‐12, at 0.15 M (KCl) ionic strength, at 25.0 ± 0.1 °C temperatures, under N2 atmosphere.SincepKa1valuefallsbelowthelowerapplicabilitylimit(<2)ofpH‐ metrictitration,thepKavalueswerealsomeasuredusingtheco‐solventmethod. (a) + H+ COO N H Lis 2- N H3 + H+ COO N O NH3 NH 2 N H COO HLis - COO N N H2 COO O H 2Lis N H3 COO N H2 N O + H+ COO COOH N H2 C OOH H 3Lis + (b) O NH3 + H+ N N O COOH H 4Lis 2+ HLis - H 2Lis 100 Lis 2- H 4Lis 2+ 80 H 3Lis + % Species 60 % Species 40 20 0 2 4 6 8 10 12 pH Figure1.16.Protonationschemeof(a)tetraproticlisinopril, (b)distributionofmacrospecies 43 Chapter1 The apparent pKa values of the COOH groups obtained in 14‐44 wt% metha‐ nol/water mixtures shifted up to the established measurable pH range, and a reliableaqueouspKa1valuecouldbeobtainedbyYSextrapolation. Fortheexactprotonspeciationoflisinopril,1HNMR/pHtitrationswithinsitupH measurementswerecarriedout,usingthemostsimilarexperimentalconditions aspossibleinpotentiometry.Thismethodwasusefultoassigntheconstantsto thefunctionalgroups:pKa1andpKa2belongtotheCOOHgroups,pKa3referstothe secondary amine (–NH–), and pKa4 shows the basicity of the primary amine (–NH2)function. The highly precise pKa values were calculated as an average of the best two valuesobtainedbyindependentmethods(Table1.7).Thesevalueswereusedto calculate the distribution curve of different protonated species of lisinopril againstthepH(Figure1.16b). Table1.7.ThepKavaluesoflisinoprilmeasuredbydifferentmethods method ionizationconstants pKa1±SD pKa2±SD pKa3±SD pKa4±SD potentiometry 1.54±0.05 3.10±0.01 7.14±0.01 10.74±0.01 potentiometryinsolventmixtures 1.62±0.01 3.21±0.02 7.22±0.03 10.75±0.01 NMR/pHtitration 1.63±0.01 3.15±0.01 7.12±0.01 10.53±0.03 averageofthebesttwovalues 1.63±0.01 3.13±0.01 7.13±0.01 10.75±0.01 1.4.2. logSdetermination Case4. Sample: hydrochlorothiazide Method: SSF Instrumentation:RadiometerPH220pHmeter;LAUDAM20Sthermostat; HeidolphMR1000magneticstirrer;JASCOV‐550UV/VIS spectrophotometer HydrochlorothiazideisabivalentacidwithpKavalues:8.75and9.88.Itsintrinsic solubility(So)valuewasmeasuredatpH6.0usingtheSSFmethod[81]. First the So of the sample was measured according to a standard (literature) protocol with the following conditions. Buffer: Britton‐Robinson (BR); solid excess:smallamount;temperature:25.0±0.1°C;equilibrationtime:48hstirring plus 24 h sedimentation; phase separation technique: sedimentation; concen‐ tration measurement: UV spectroscopy (λ=271 nm, A1%1cm: 696); number of parallels:6.Result:So=556±13.2μg/ml. Next,differentparametersofthisprotocolwereexamined,alwaysoneofthesix parameters(bufferchoice,amountofsolidexcess,temperature,timeofstirring, time of sedimentation, phase separation technique) was varied while the other conditionswerekeptunchanged. 44 Physicochemicalprofilingindrugresearchanddevelopment Effect of buffer solution.Three buffer solutions were used at pH 6.0. The results areshowninFigure1.17a.ThestatisticalanalysishasindicatedthatSovaluesin BR and Sörensen phosphate (I) buffers are in accordance, but the solubility in Sörensencitrate(II)bufferdeviatessignificantly.Theionicstrengthofthislater bufferisfourtimeshigherthanthatofBRorSörensenI. Effect of phase separation. Alternative techniques to sedimentation such as cen‐ trifugation and filtration were studied. 12 samples were centrifuged after 48h stirringat2000rpmfor10min,while12sampleswerefilteredthrough0.45μm membrane filters. Results shown in Figure 1.17b are significantly different. The highestdeviationiscausedbyfiltration. Effectofequilibrationtime.Figure1.17cshowstheexperimentalresultsobtained when(i)stirringtimewaschangedfrom30minto48hfollowedbya24hsedi‐ mentation;and(ii)sedimentationtimewaschangedfrom1hto24h,keepingthe stirringtimeconstant(48h).Fromtheresultsitcanbeconcludedthatthetimeof sedimentation plays a greater role in the development of equilibrium than the timeofintensiveagitation. Effect of temperature. The solubility of hydrochlorothiazide increases with the temperature (Figure 1.17d). It is almost double at 37 °C than at 25 °C, which underlines the need for solubility determination at biomimetic temperature as well. (a) (b) 779 g / ml 600 580 560 , Solubility 540 516 520 500 480 460 0.5 Solubility,g / ml 565 Sörensen I. 564 508 1 563 661 591 556 sedimentation centrifugation 12 24 48 Time [h] Time, h g / ml 640 631 610 610 631 610 620 605 606 600 , Solubility 575 580 580 556 560 540 520 500 1 2 4 6 8 12 18 24 Solubility,g / ml Time, h Time [h] filtration 556 1200 6 (d) 580 523 2 680 660 640 620 600 580 560 540 520 500 Sörensen II. 1000 g / ml 800 , Solubility 600 Solubility,g / ml (c) Solubility,g / ml Solubility,g / ml 900 800 700 g / ml 556 600 Solubility , 500 400 300 200 100 0 Britton-Robinson 1036 556 450 400 200 0 15 °C 25 °C 37 °C Figure1.17.Effectofexperimentalconditionsontheintrinsicequilibriumsolubilityof hydrochlorothiazide:(a)buffersolution,(b)phaseseparationtechnique, (c)stirringtime(uppergraph)sedimentationtime(lowergraph),(d)temperature 45 Chapter1 Case5. Sample: papaverinehydrochloride Method: SSF Instrumentation: RadiometerPH220pHmeter;LAUDAM20Sthermostat; HeidolphMR1000magneticstirrer;JASCOV‐550UV/VIS spectrophotometer Thesolubility‐pHprofileofpapaverinehydrochloridewasdeterminedinawide pHrangeusinganew(shorter)protocolderivedfromtheSSFmethod:BRbuffer, smallsolidexcess,25.0±0.1°C,6hstirringand18hsedimentation[42]. First,theintrinsicsolubilityofthesamplewasmeasuredathighpH(11.71)and found 17 μg/ml (log So = 1.70 [log μM]). Then the equilibrium solubility (SpH) valuesattwelvedifferentpHvaluesbetween0.06and8.02weredetermined. FromlogSoandpKa(6.36)valuesthetheoreticallogSpH/pHprofilwasgenerated by the HH equation. Figure 1.18 shows the excellent agreement between the experimentaldatapointsandthepredictedHHcurve.Thisshapeistypicalfora monovalentbase,wherethesolubilityincreaseswithadecreaseofthepH,asthe freebasestartstoconverttotheprotonatedform.AtaroundpH3,thesolubility of the papaverine hydrochloride salt reaches the maximum (pHmax), which is limitedbythesolubilityproduct.BetweenpH2and3thereisaconstantvaluefor the salt solubility. Below pH 2 the solubility of the salt decreases due to the commonioneffect,causedbychlorideionsfromHClusedtoadjustthepH. Figure1.18.Solubility‐pHprofileofpapaverinehydrochloride 46 Physicochemicalprofilingindrugresearchanddevelopment This example proves that the HH equation can be used for the calculation of solubility at physiological important pH values once the intrinsic solubility and thepKavaluehavebeenpreciouslydetermined[42]. Case6. Sample: telmisartan Method: SSF Instrumentation: RadiometerPH220pHmeter;LAUDAthermostat;Heidolph MR1000magneticstirrer;JASCOV‐550UV/VIS spectrophotometer Thesolubilityoftelmisartanwasmeasuredindistilledwater(ordinarilypH~6) andat37±0.1°Ctemperature(oneoftheconditionswheresolubilityisrequired bytheregistrationauthorities).Solidmaterialat0.01gwasaddedto20mlfresh‐ lyboiledandcooledwaterandthenthenew(shorter)protocolderivedfromthe SSFmethodwasfollowed.Aliquotsweretakenoutfromthesupernatantandthe absorbance was measured without dilution at λ = 295 nm, in a cell with a 5cm pathlength.TheconcentrationwascalculatedusingA1%1cm=510measuredsepa‐ rately prior to solubility measurement. From three parallel experiments, the solubility of telmisartan was found as low as SpH=0.50±0.09μg/ml. The relati‐ velyhigherror(SD=±18%)isduetotheverylowsolubility(thelowestvalue wecouldevermeasurebytheSSFmethod)andtheoccasionallyformedsupersa‐ turated solution, from which small (invisible) particles precipitated in the cell uponabsorbancemeasurement. Case7. Sample: maprotiline Method: SSFandCheqSol Instrumentation: RadiometerPH220pHmeter;LAUDAthermostat;Heidolph MR1000magneticstirrer;JASCOV‐550UV/VIS spectrophotometerandGLpKa+D‐PAS The precise intrinsic solubility of maprotiline base (pKa= 10.33) ‐ another very sparingly soluble compound ‐ could not be determined by the SSF method. The resultobtainedfromthreeseparate measurementsinBR bufferatpH11.5was So=8.05±3μg/ml. The reason for the extremely high experimental error (SD=±37%) is that a colloid, slightly opalescent solution (perhaps due to re‐ crystallization or supersaturation) was formed upon equilibration. This opale‐ scence could be eliminated by neither filtration nor centrifugation. So, the SSF solubility result must be considered as an approximate value. Thus, the poten‐ tiometricmethod,namelytheChasingEquilibriumSolubility(CheqSol)wasalso applied.Maprotilinewasadded(2mg)to10mlof0.15MKClsolutionthenpre‐ acidified with 0.5 M HCl to pH 2 where the compound was fully dissolved. This solution was titrated with 0.5 M KOH until the solution became cloudy, which indicatedtheprecipitationofthefreebaseform.Theoccurrenceofprecipitation was detected using a spectroscopic dip probe then the solution was quickly 47 Chapter1 brought close to equilibrium by adding very small amounts of acidic or basic titrants alternatively resulting in an oscillation between supersaturation and subsaturation. The Bjerrum plot of titration is shown in Figure 1.19. While the sample is fully dissolved, the experimental data fit well to the nonprecipitation theoreticalcurve(a).Afterprecipitation,thepointslieclosetotheprecipitation theoretical curve (b). The precipitation point is used to calculate the kinetic solubilityvalue.Theintrinsicequilibriumsolubilitywasdeterminedfrom40data pointswith8zeropHgradientcrossings. The intrinsic solubility was obtained as average of 6 separate titrations, So=5.8±0.3μg/ml.ThelowSDindicatesthehigherprecisionofthedataandthe advantageoftheCheqSolmethodinthiscase. Figure1.19.BjerrumplotofsolubilitydeterminationofmaprotilinebyCheqSolmethod. (a)nonprecipitationtheoreticalcurve,(b)precipitationtheoreticalcurve Case8. Sample: venlafaxineHCl Method: SSF Instrumentation: RadiometerPH220pHmeter;LAUDAM20Sthermostat; HeidolphMR1000magneticstirrer;JASCOV‐550UV/VIS spectrophotometer Venlafaxineisamonovalentbase(pKa=9.6),itshydrochloridesaltcanformdif‐ ferent polymorphs. The solubility of two polymorph forms (I and II) was inves‐ tigatedatthreepHvalues:4.9(unadjustedpHindistilledwater),8.9(BRbuffer), and12(0.001MNaOH)at37±0.1°CtemperatureusingtheSSFmethod.There‐ sults are summarized in Table 1.8. The salt solubility is higher than 50 % (g/100ml)inthecaseofbothpolymorphs.Theintrinsicsolubilityofvenlafaxine 48 Physicochemicalprofilingindrugresearchanddevelopment measured at pH 12 was also found to be the same for both I and II forms (So=460±10μg/ml). Thediffractionanalysisofthesolidphasefilteredoutattheendofthesolubility measurement revealed that polymorphs I and II equally converted to the same crystal form of free base venlafaxine. This experience underscores the need for analysisofthesolidphaseaftertheequilibriumstatehasbeenreached. Table1.8.Solubility(g/100ml)oftwopolymorphformsofvenlafaxine hydrochlorideatthreepHvaluesandat37°Ctemperature venlafaxinehydrochloride SpH(pH4.9) SpH(pH8.9) So(pH12.0) FormI >50 0.180 0.046 FormII >50 0.208 0.046 1.4.3. logPdetermination Case9. Sample: chlorpromazine Method: SSF Instrumentation: LAUDAM20Sthermostat;Hawlett‐Packard8452AUV/VIS spectro‐photometer Chlorpromazine is a very lipophilic monovalent base (pKa: 9.24). The true logP valuecannotbemeasuredathighpHvalues(>11.5)directlybytheSFmethod becauseofthelowsolubilityofthefreebaseformofthecompoundathighpH.In suchcases(whichistypicalamongdrugs),thelogDpHismeasuredatdifferentpH valuesatwhichthemoleculepartiallyionizesanddissolvesbetterandthenitis converted to the true log P using Equation 1.30b. The log DpH values of chlorpromazineweremeasuredatthreepHvalues(7.4,8.0and8.5)inBRbuffer astheaqueousphase,usinganR=200and100phaseratios(50mlbuffer:0.25 mloctanoland25mlbuffer:0.25mloctanol).Wefollowedthestandardprotocol oftheSFmethod:1hintensiveshakinginashakingthermostat;phaseseparation bycentrifugation(730gfor10min).Theabsorbanceoftheaqueousphasebefore (Ao) and after (A1) the partition was measured by spectroscopy at λ = 254 nm. The apparent partition coefficient is calculated according to DpH = [(Ao‐A1)/A1]R [104].Thelipopilicity‐pHprofileisshowninFigure1.20. Result:logP=5.13±0.10(n=18) 49 Chapter1 Figure1.20.Lipophilicity‐pHprofileofchlorpromazine (pointsrepresenttheexperimentallymeasuredlogDpHvalues) Case10. Sample: deramciclane Method: potentiometricmethod Instrumentation: PCA101(Sirius,UK) Deramciclane was an original anxiolytic molecule developed by EGIS (Hungary) inthelate‘90s,whichunfortunatelyfailedfromclinicalphaseIII.Itisasparingly solublemonovalentbase(pKa=9.61).IthasaveryweakUVabsorption(lowspe‐ cificabsorptivity)thuslipophilicitydeterminationbytheSFmethod(asdonein Case9)ishindered.ThelogPvaluewasmeasuredbydual‐phasepotentiometric titrationat25.0± 0.1°C temperature,underN2atmosphere.Sixtitrations were performedbetweenpH3and12,ina1mMconcentrationsolutionofthesample using 15 ml water and 0.05 ml octanol phase (Figure 1.21a). From these titrations the apparent pKa values (measured in the presence of octanol, poKa) wereobtained.TheBjerrumplot(Figure1.21b)showsbigshifttowardlowerpH values(typicalforbases)whichindicateshighlipophilicityofthesample.Thelog P value is calculated according to the equation: log P = (10(pKa ‐ poKa) – 1)/r. The extreme(octanol/water)phaseratio(r=0.0033)usedhereallowedlipophilicity measurementashighaslogP=5.90±0.02(n=6).Accordingtoourexperiences, this represents the upper limit of the pH‐metric log P determination method [113]. 50 Physicochemicalprofilingindrugresearchanddevelopment Figure1.21.pH‐metriclogPdeterminationofderamciclane: (a)titrationcurvesinthepresenceofdifferentamountsofoctanol,(b)Bjerrumplot Case11. Sample: prostaglandinE1‐ethylester(PGEE) Method: RP‐TLC Instrumentation: RP‐diC1silanizedplates,Merck#5747;Camag microsampler;ShimadzuCS‐9301CPdensitometer PGEE is an example for molecules where classical, standard methods cannot be applied. Due to the lack of useful UV absorption (above λ > 230 nm) or an ionizablegroup,neithertheSFnorpH‐metrycanbeused.LogPwasdetermined byavalidatedRP‐TLCmethod. O COOC2H5 CH3 HO OH Measurementwasperformedon20cmx20cmplatesprecoatedwithsilanized silicagelGF254asthestationaryphaseandmethanol/water(55:45)asthemobile phase. Before use, the plates were washed with methanol (ascending deve‐ lopment),thendriedandheatedat160°Cfor1h.Thesamples(PGEEandcali‐ bration set) were dissolved in a 1 : 1 methanol/chloroform mixture (2mg/ml) and 2 μl was spotted on the plate. The chamber was saturated with the mobile phase for 30 min before use. After development the plates were dried and evaluatedbydensitometry. The calibration curve was set up using seven compounds [114] and obtained fromthreeparallelruns:logP=3.508RM+0.968(r=0.995,n=21).ThelogPof PGEEwascalculatedwiththehelpofthisequation. Result:logPTLC=4.02±0.05(n=3). 51 Chapter1 1.5. OUTLOOK Concerning the role of physicochemical profiling in the future, we can certainly predict that it remains an integrated part of drug research providing a simple, cheap,andfasttoolfortheestimationofADMETparametersintheearlystageof DD&D. A higher level of automation (e.g. integration of several robotic platforms) and highersensitivityofdetectionmethodscanbeexpectedleadingtotheincreaseof the HT feature of the applied methods, but it must be synchronous with the improvement of the reliability of the data determined. Next to this, the cost‐ effectivenesswillbethecriticalfactorintheselectionbetweenmethodshaving thesamecapacity. We can anticipate the increasing application of biorelevant experimental conditions in physicochemical profiling. Standardization and validation of these biomimeticsystemsareobviouslynecessaryinthenearfuture. The use of in silico methods will be growing if further development of computational approaches results in even more reliable data. For the in silico methodsbasedonbigdatabasesthe qualityoftheinputofexperimentalvalues mustbefurtherimproved. Finally,moreeffectiveusageofphysicochemicalprofilingindrugresearchcanbe promoted by including informative courses or seminars, for example, in higher educationtostrengthenthisspecialfieldofmedicinalchemistryinacademia. Acknowledgement IwouldliketothankmycolleagueGergelyVölgyi,PhDforhisexperimentalwork, valuablesuggestions,andhelpinpreparingthefigures.IalsothanktheHungarian NationalScienceFoundation(GrantNo.:OTKAK78102)forfinancialsupport. REFERENCES 1. J. Wang, L. Urban. The impact of early ADME profiling on drug discovery and developmentstrategy.DrugDiscoveryWorldFall(2004)73‐86. 2. C. Hansch, P.P. Maloney, T. Fujita, R. Muir. Correlation of biological activity of phenoxyacetic acids with Hammett substituent constants and partition coefficients.Nature194(1962)178‐180. 3. G.K.Dixon,J.P.Major,M.J.Rice.,HighThroughputScreening:TheNextGeneration Bios,Oxford,2000. 4. K. 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